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Hydrogels are widely used as hydrated matrices for cell encapsulation in a number of applications, spanning from advanced 3D cultures and tissue models to cell-based therapeutics and tissue engineering. Hydrogel formation in the presence of living cells requires cross-linking reactions that proceed efficiently under close to physiological conditions. Recently, the nucleophilic aromatic substitution of phenyl-oxadiazole (Ox) methylsulfones (MS) by thiols was introduced as a new cross-linking reaction for cell encapsulation. Reported poly(ethylene glycol) (PEG)-based hydrogels featured tunable gelation times within seconds to a few minutes within pH 8.0 to 6.6 and allowed reasonably good mixing with cells. However, their rapid degradation prevented cell cultures to be maintained beyond 1 week. In this Article, we present the reactivity optimization of the heteroaromatic ring of the MS partner to slow down the cross-linking kinetics and the degradability of the derived hydrogels. New MS substrates based on phenyl-tetrazole (Tz) and benzothiazole (Bt) rings, with lower electrophilicity than Ox, were synthesized by simple pathways. When mixed with PEG-thiol, the novel PEG-MS extended the working time of precursor mixtures and allowed longer term cell culture. The Tz-based MS substrate was identified as the best candidate, as it is accessible by simple chemical reactions from cost-effective reactants, hydrogel precursors show good stability in aqueous solution and keep high chemoselectivity for thiols, and the derived Tz gels support cell cultures for >2 weeks. The Tz system also shows tunable gelation kinetics within seconds to hours and allows comfortable manipulation and cell encapsulation. Our findings expand the toolkit of thiol-mediated chemistry for the synthesis of hydrogels with improved properties for laboratory handling and future automatization.
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Encapsulación Celular , Hidrogeles , Técnicas de Cultivo de Célula , Polietilenglicoles , Compuestos de SulfhidriloRESUMEN
Neuro-regeneration after trauma requires growth and reconnection of neurons to reestablish information flow in particular directions across the damaged tissue. To support this process, biomaterials for nerve tissue regeneration need to provide spatial information to adhesion receptors on the cell membrane and to provide directionality to growing neurites. Here, photoactivatable adhesive peptides based on the CASIKVAVSADR laminin peptidomimetic are presented and applied to spatiotemporal control of neuronal growth to biomaterials in vitro. The introduction of a photoremovable group [6-nitroveratryl (NVOC), 3-(4,5-dimethoxy-2-nitrophenyl)butan-2-yl (DMNPB), or 2,2'-((3'-(1-hydroxypropan-2-yl)-4'-nitro-[1,1'-biphenyl]-4-yl)azanediyl)bis(ethan-1-ol) (HANBP)] at the amino terminal group of the K residue temporally inhibited the activity of the peptide. The bioactivity was regained through controlled light exposure. When used in neuronal culture substrates, the peptides allowed light-based control of the attachment and differentiation of neuronal cells. Site-selective irradiation activated adhesion and differentiation cues and guided seeded neurons to grow in predefined patterns. This is the first demonstration of ligand-based light-controlled interaction between neuronal cells and biomaterials.
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Materiales Biocompatibles/farmacología , Neurogénesis/efectos de los fármacos , Neurogénesis/efectos de la radiación , Neuronas/efectos de los fármacos , Neuronas/efectos de la radiación , Péptidos/farmacología , Secuencia de Aminoácidos , Animales , Materiales Biocompatibles/química , Adhesión Celular/efectos de los fármacos , Adhesión Celular/efectos de la radiación , Células Cultivadas , Laminina/química , Laminina/farmacología , Ligandos , Ratones Endogámicos C57BL , Neuronas/citología , Péptidos/química , FotólisisRESUMEN
The exploitation of easily accessible and nontoxic natural catechol compounds for surface functionalization and coating is attracting growing interest for biomedical applications. We report herein the deposition on different substrates of chemically stable thin films by autoxidation of 1 mM caffeic acid (CA) solutions at pH 9 in the presence of equimolar amounts of hexamethylenediamine (HMDA). UV-visible, mass spectrometric, and solid state 13C and 15N NMR analysis indicated covalent incorporation of the amine during CA polymerization to produce insoluble trioxybenzacridinium scaffolds decorated with carboxyl and amine functionalities. Similar coatings are obtained by replacing CA with 4-methylcatechol (MC) in the presence of HMDA. No significant film deposition was detected in the absence of HMDA nor by replacing it with shorter chain ethylenediamine, or with monoamines. The CA/HMDA-based films resisted oxidative and reductive treatments, displayed efficient Fe(II) and Cu(II) binding capacity and organic dyes adsorption, and provided an excellent cytocompatible platform for growing embryonic stem cells. These results pointed to HMDA as an efficient cross-linking mediator of film deposition from natural catechols for surface functionalization and coatings.
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Biomaterials for cell culture allowing simple and quantitative presentation of instructive cues enable rationalization of the interplay between cells and their surrounding microenvironment. Poly(acrylamide) (PAAm) hydrogels are popular 2D-model substrates for this purpose. However, quantitative and reproducible biofunctionalization of PAAm hydrogels with multiple ligands in a trustable, controlled, and independent fashion is not trivial. Here, we describe a method for bifunctional modification of PAAm hydrogels with thiol- and amine- containing biomolecules with controlled densities in an independent, orthogonal manner. We developed copolymer networks of AAm with 9% acrylic acid and 2% N-(4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)acrylamide. The covalent binding of thiol- and amine-containing chromophores at tunable concentrations was demonstrated and quantified by UV spectroscopy. The morphology, mechanical properties, and homogeneity of the copolymerized hydrogels were characterized by scanning electron microscopy, dynamic mechanical analysis, and confocal microscopy studies. Our copolymer hydrogels were bifunctionalized with polylysine and a laminin-mimetic peptide using the specific chemistries. We analyzed the effect of binding protocol of the two components in the maturation of cultured postmitotic cortical neurons. Our substrates supported neuronal attachment, proliferation, and neuronal differentiation. We found that neurons cultured on our hydrogels bifunctionalized with ligand-specific chemistries in a sequential fashion exhibited higher maturation at comparable culture times than using a simultaneous bifunctionalization strategy, displaying a higher number of neurites, branches, and dendritic filopodia. These results demonstrate the relevance of quantitative and optimized coupling chemistries for the performance of simple biomaterials and with sensitive cell types.
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Resinas Acrílicas/química , Materiales Biocompatibles/química , Hidrogeles/química , Animales , Células Cultivadas , Laminina/química , Ratones , Ratones Endogámicos C57BL , Polilisina/química , Polímeros/químicaRESUMEN
Materials engineered to elicit targeted cellular responses in regenerative medicine must display bioligands with precise spatial and temporal control. Although materials with temporally regulated presentation of bioadhesive ligands using external triggers, such as light and electric fields, have recently been realized for cells in culture, the impact of in vivo temporal ligand presentation on cell-material responses is unknown. Here, we present a general strategy to temporally and spatially control the in vivo presentation of bioligands using cell-adhesive peptides with a protecting group that can be easily removed via transdermal light exposure to render the peptide fully active. We demonstrate that non-invasive, transdermal time-regulated activation of cell-adhesive RGD peptide on implanted biomaterials regulates in vivo cell adhesion, inflammation, fibrous encapsulation, and vascularization of the material. This work shows that triggered in vivo presentation of bioligands can be harnessed to direct tissue reparative responses associated with implanted biomaterials.
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Adhesión Celular/efectos de los fármacos , Erupciones por Medicamentos/inmunología , Fibroblastos/inmunología , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/inmunología , Oligopéptidos/efectos adversos , Oligopéptidos/efectos de la radiación , Animales , Materiales Biocompatibles/química , Adhesión Celular/inmunología , Adhesión Celular/efectos de la radiación , Moléculas de Adhesión Celular/efectos adversos , Moléculas de Adhesión Celular/efectos de la radiación , Línea Celular , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Humanos , Luz , Masculino , Ratones , Ratones Endogámicos BALB C , Células 3T3 NIHRESUMEN
Poly(acrylamide) P(AAm) gels have become relevant model substrates to study cell response to the mechanical and biochemical properties of the cellular microenvironment. However, current bioconjugation strategies to functionalize P(AAm) gels, mainly using photoinduced arylazide coupling, show poor specificity and hinder conclusive studies of material properties and cellular responses. We describe methylsulfonyl-containing P(AAm) hydrogels for cell culture. These gels allow easy, specific and functional covalent coupling of thiol containing bioligands in tunable concentrations under physiological conditions, while retaining the same swelling, porosity, cytocompatibility, and low protein adsorption of P(AAm) gels. These materials allow quantitative and standardized studies of cell-materials interactions with P(AAm) gels.
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Resinas Acrílicas/química , Técnicas de Cultivo de Célula , Hidrogeles/química , Hidrogeles/síntesis química , Compuestos de Sulfhidrilo/química , Células HeLa , Humanos , Estructura Molecular , Células Tumorales CultivadasRESUMEN
The curing time of an adhesive material is determined by the polymerization and cross-linking kinetics of the adhesive formulation and needs to be optimized for the particular application. Here, we explore the possibility of tuning the polymerization kinetics and final mechanical properties of tissue-adhesive PEG gels formed by polymerization of end-functionalized star-PEGs with catecholamines with varying substituents. We show strong differences in cross-linking time and cohesiveness of the final gels among the catecholamine-PEG variants. Installation of an electron-withdrawing but π-electron donating chloro substituent on the catechol ring resulted in faster and more efficient cross-linking, while opposite effects were observed with the strongly electron-withdrawing nitro group. Chain substitution slowed down the kinetics and hindered cross-linking due either to chain breakdown (ß-OH group, in norepinephrine) or intramolecular cyclization (α-carboxyl group, in DOPA). Interesting perspectives derive from use of mixtures of catecholamine-PEG precursors offering further opportunities for fine-tuning of the curing parameters. These are interesting properties for the application of catecholamine-PEG gels as tissue glues or biomaterials for cell encapsulation.
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Materiales Biocompatibles/química , Catecolaminas/química , Catecoles/química , Reactivos de Enlaces Cruzados/química , Polietilenglicoles/química , Adhesivos Tisulares/química , Dihidroxifenilalanina/química , Geles , Cinética , Norepinefrina/química , Polimerizacion , Relación Estructura-Actividad , Ingeniería de TejidosRESUMEN
Surface topography and work function maps were simultaneously obtained for carbon surfaces modified by a dendritic molecule: 3,5-Bis (3,5-dinitrobenzoylamino) benzoic acid. The dendrons were spontaneously assembled onto highly ordered pyrolytic graphite samples, exhibiting an increase in the surface potential. This fact is consistent with the incorporation of an electron-acceptor functional group that remains electroactive on the surface.
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Benzoatos/química , Dendrímeros/química , Grafito/química , Microscopía de Fuerza Atómica , Nanoestructuras/química , Propiedades de SuperficieRESUMEN
Injectable hydrogels have emerged as intelligent and versatile materials that have been proven to possess huge potential for many biomedical applications including drug delivery, tissue engineering, and regenerative medicine. Hydrogels are a class of polymers with highly hydrated 3D networks that have microenvironmental properties such as oxygen/nutrient permeability that are similar to the native extracellular matrix. In addition to possessing the typical advantages of conventional hydrogels, injectable hydrogels offer extra unique features, enabling minimally invasive injectability and durability for irregularly shaped sites, and the possibility of processing these materials via, e.g., additive manufacturing techniques. As such, there has been a growing interest in using injectable hydrogels as scaffolds/carriers for therapeutic agents, including but not limited to drugs, cells, proteins, and bioactive molecules, targeted to treat chronic diseases including cancer, but also to facilitate the repair and regeneration of damaged organs/tissues. In this themed collection of Journal of Materials Chemistry B and Biomaterials Science, we include outstanding contributions covering recent developments in this rapidly evolving field of injectable hydrogels including emerging chemistries, synthesis pathways, fabrication methods, cell-material interaction, in vitro, ex vivo and in vivo performances, and subsequent targeted applications (drug delivery, tissue engineering and regenerative medicine) of injectable hydrogels.
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Materiales Biocompatibles , Hidrogeles , Inyecciones , Ingeniería de Tejidos , Hidrogeles/química , Humanos , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Materiales Biocompatibles/síntesis química , Medicina Regenerativa/métodos , Sistemas de Liberación de Medicamentos , AnimalesRESUMEN
Hydrogel precursors that crosslink within minutes are essential for the development of cell encapsulation matrices and their implementation in automated systems. Such timescales allow sufficient mixing of cells and hydrogel precursors under low shear forces and the achievement of homogeneous networks and cell distributions in the 3D cell culture. The previous work showed that the thiol-tetrazole methylsulfone (TzMS) reaction crosslinks star-poly(ethylene glycol) (PEG) hydrogels within minutes at around physiological pH and can be accelerated or slowed down with small pH changes. The resulting hydrogels are cytocompatible and stable in cell culture conditions. Here, the gelation kinetics and mechanical properties of PEG-based hydrogels formed by thiol-TzMS crosslinking as a function of buffer, crosslinker structure and degree of TzMS functionality are reported. Crosslinkers of different architecture, length and chemical nature (PEG versus peptide) are tested, and degree of TzMS functionality is modified by inclusion of RGD cell-adhesive ligand, all at concentration ranges typically used in cell culture. These studies corroborate that thiol/PEG-4TzMS hydrogels show gelation times and stiffnesses that are suitable for 3D cell encapsulation and tunable through changes in hydrogel composition. The results of this study guide formulation of encapsulating hydrogels for manual and automated 3D cell culture.
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Encapsulación Celular , Compuestos de Sulfhidrilo , Compuestos de Sulfhidrilo/química , Hidrogeles/química , Cinética , Técnicas de Cultivo de Célula/métodos , Materiales Biocompatibles/química , Polietilenglicoles/químicaRESUMEN
Adv. Sci. 2019, 6, 1801982 DOI: 10.1002/advs.201801982 The above article, published online on May 3, 2019, in Wiley Online Library (https://doi.org/10.1002/advs.201801982), has been retracted by agreement between the authors, the journal Editor-in-Chief Kirsten Severing, and Wiley-VCH GmbH. The retraction has been agreed on following concerns raised by a third party and a subsequent investigation by the corresponding authors. Data depicted in Figure 4 and Figure 5 could not be reproduced in follow-up experiments. Therefore, the conclusions associated with those figures in the article are considered invalid. E.S.K. participated in the study design, performed measurements, analyzed the data, compiled the figures and participated in manuscript writing. A.d.C. and S.S. participated in the study design, research supervision, and manuscript writing. J.I.P. participated in the study design. M.K.L.H. participated in research supervision and manuscript revision. C.M. assisted with the experimental procedures and data collection.
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There is an increasing interest in coupling reactions for cross-linking of cell-encapsulating hydrogels under biocompatible, chemoselective, and tunable conditions. Inspired by the biosynthesis of luciferins in fireflies, here we exploit the cyanobenzothiazole-cysteine (CBT-Cys) click ligation to develop polyethylene glycol hydrogels as tunable scaffolds for cell encapsulation. Taking advantage of the chemoselectivity and versatility of CBT-Cys ligation, a highly flexible gel platform is reported here. We demonstrate luciferin-inspired hydrogels with important advantages for cell encapsulation applications: (i) gel precursors derived from inexpensive reagents and with good stability in aqueous solution (>4 weeks), (ii) adjustable gel mechanics within physiological ranges (E = 180-6240 Pa), (iii) easy tunability of the gelation rate (seconds to minutes) by external means, (iv) high microscale homogeneity, (v) good cytocompatibility, and (iv) regulable biological properties. These flexible and robust CBT-Cys hydrogels are proved as supportive matrices for 3D culture of different cell types, namely, fibroblasts and human mesenchymal stem cells. Our findings expand the toolkit of click chemistries for the fabrication of tunable biomaterials.
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Materiales Biocompatibles/química , Técnicas de Cultivo Tridimensional de Células , Hidrogeles/química , Luciferinas/química , Células Cultivadas , Humanos , Ensayo de Materiales , Estructura Molecular , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Hydrogel biomaterials in combination with living cells are applied in cell biology, tissue engineering and regenerative medicine. In particular, poly(acrylamide) (PAM) hydrogels are frequently used in cell biology laboratories as soft substrates for 2D cell culture. These biomaterials present advantages such as the straightforward synthesis, regulable mechanical properties within physiological range of native soft tissues, the possibility to be biofunctionalized with ligands to support the culture of living cells, and their optical transparency that makes them compatible with microscopy methods. Due to the chemical inertness and protein repellant properties of PAM hydrogels, these materials alone do not support the adhesion of cells. Therefore, biofunctionalization of PAM gels is necessary to confer them bioactivity and to promote cell-material interactions. Herein, the current chemical strategies for the bioconjugation of ligands to PAM gels are reviewed. Different aspects of the existing bioconjugation methods such as chemo-selectivity and site-specificity of attachment, preservation of ligand's functionality after binding, user-friendliness and cost are presented and compared. This work aims at guiding users in the choice of a strategy to biofunctionalize PAM gels with desired biochemical properties.
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Osteoarthritis (OA) and chronic low back pain due to degenerative (intervertebral) disc disease (DDD) are two of the major causes of disabilities worldwide, affecting hundreds of millions of people and leading to a high socioeconomic burden. Although OA occurs in synovial joints and DDD occurs in cartilaginous joints, the similarities are striking, with both joints showing commonalities in the nature of the tissues and in the degenerative processes during disease. Consequently, repair strategies for articular cartilage (AC) and nucleus pulposus (NP), the core of the intervertebral disc, in the context of OA and DDD share common aspects. One of such tissue engineering approaches is the use of injectable hydrogels for AC and NP repair. In this review, the state-of-the-art and recent developments in injectable hydrogels for repairing, restoring, and regenerating AC tissue suffering from OA and NP tissue in DDD are summarized focusing on cell-free approaches. The various biomaterial strategies exploited for repair of both tissues are compared, and the synergies that could be gained by translating experiences from one tissue to the other are identified. Impact statement Joints affected by osteoarthritis (OA) and degenerative (intervertebral) disc disease (DDD) share similarities in tissue composition and in the degenerative disease processes. This has led to the development of similar tissue engineering approaches to repair the articular cartilage (AC) and the nucleus pulposus (NP), in the context of OA and DDD, such as injectable hydrogels. In this review, recent developments in injectable hydrogels for repair of AC and NP tissues are summarized, biomaterial strategies are compared, and synergies are identified focusing on cell-free approaches. The summarized developments are expected to inspire more cross talk between both research fields.
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Cartílago Articular , Degeneración del Disco Intervertebral , Disco Intervertebral , Núcleo Pulposo , Osteoartritis , Materiales Biocompatibles , Humanos , Hidrogeles/farmacología , Desplazamiento del Disco Intervertebral , Osteoartritis/terapiaRESUMEN
Hydrogels for wound management and tissue gluing applications have to adhere to tissues for a given time scale and then disappear, either by removal from the skin or by slow degradation for applications inside the body. Advanced wound management materials also envision the encapsulation of therapeutic drugs or cells to support the natural healing process. The design of hydrogels that can fulfill all of these properties with minimal chemical complexity, a stringent condition to favor transfer into a real medical device, is challenging. Herein, we present a hydrogel design with a moderate structural complexity that fulfills a number of relevant properties for wound dressing: it can form in situ and encapsulate cells, it can adhere to tissues, and it can be degraded on demand by light exposure under cytocompatible conditions. The hydrogels are based on starPEG macromers terminated with catechol groups as cross-linking units and contain intercalated photocleavable nitrobenzyl triazole groups. Hydrogels are formed under mild conditions (N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid (HEPES) buffer with 9-18 mM sodium periodate as the oxidant) and are compatible with encapsulated cells. Upon light irradiation, the cleavage of the nitrobenzyl group mediates depolymerization, which enables the on-demand release of cells and debonding from tissues. The molecular design and obtained properties reported here are interesting for the development of advanced wound dressings and cell therapies and expand the range of functionality of current alternatives.
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Hidrogeles/química , Luz , Animales , Adhesión Celular , Técnicas de Cultivo de Célula , Encapsulación Celular , Línea Celular , Hidrogeles/metabolismo , Hidrogeles/farmacología , Cinética , Ratones , Oligopéptidos/química , Oligopéptidos/metabolismo , Fotólisis/efectos de la radiación , Polietilenglicoles/química , Piel/efectos de los fármacos , Piel/metabolismo , Triazoles/químicaRESUMEN
In this paper we explore the printability of reversible networks formed by catechol functionalized PEG solutions and metal cations (Al3+, Fe3+ or V3+). The printability and shape fidelity were dependent on the ink composition (metal ion type, pH, PEG molecular weight) and printing parameters (extrusion pressure and printing speed). The relaxation time, recovery rate and viscosity of the inks were analyzed in rheology studies and correlated with thermodynamic and ligand exchange kinetic constants of the dynamic bonds and the printing performance (i.e. shape fidelity of the printed structures). The relevance of the relaxation time and ligand exchange kinetics for printability was demonstrated. Cells seeded on the materials crosslinked with Al3+, Fe3+ ions were viable and revealed well-spread morphologies during 7 day culture, indicating the potential of the formulations to be used as inks for cell encapsulation. The proposed dynamic ink design offers significant flexibility for 3D bioprinting, and enables straightforward adjustment of the printable formulation to meet application-specific needs.
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Bioimpresión , Catecoles/química , Reactivos de Enlaces Cruzados/química , Tinta , Polietilenglicoles/química , Animales , Línea Celular , Proliferación Celular , Supervivencia Celular , Fibroblastos/citología , Concentración de Iones de Hidrógeno , Iones , Cinética , Ligandos , Metales/química , Ratones , Impresión Tridimensional , Reología , Termodinámica , Factores de TiempoRESUMEN
Thiol-maleimide and thiol-vinylsulfone cross-linked hydrogels are widely used systems in 3D culture models, in spite of presenting uncomfortable reaction kinetics for cell encapsulation: too fast (seconds for thiol-maleimide) or too slow (minutes-hours for thiol-vinylsulfone). Here, we introduce the thiol-methylsulfone reaction as alternative cross-linking chemistry for cell encapsulation, particularized for PEG-hydrogels. The thiol-methylsulfone reaction occurs at high conversion and at intermediate reaction speed (seconds-minutes) under physiological pH range. These properties allow easy mixing of hydrogel precursors and cells to render homogeneous cell-laden gels at comfortable experimental time scales. The resulting hydrogels are cytocompatible and show comparable hydrolytic stability to thiol-vinylsulfone gels. They allow direct bioconjugation of thiol-derivatized ligands and tunable degradation kinetics by cross-linking with degradable peptide sequences. 3D cell culture of two cell types, fibroblasts and human umbilical vein endothelial cells (HUVECs), is demonstrated.
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Técnicas de Cultivo de Célula/métodos , Encapsulación Celular/métodos , Dimetilsulfóxido/química , Hidrogeles/química , Maleimidas/química , Compuestos de Sulfhidrilo/química , Sulfonas/química , Movimiento Celular , Supervivencia Celular , Dimetilsulfóxido/toxicidad , Fibroblastos/citología , Gelatina/química , Células Endoteliales de la Vena Umbilical Humana , Humanos , Hidrogeles/toxicidad , Polietilenglicoles/química , Reología , Esferoides Celulares , Sulfonas/toxicidadRESUMEN
Collagen is the most abundant structural protein in mammals and is crucial for the mechanical integrity of tissues. Hsp47, an endoplasmic reticulum resident collagen-specific chaperone, is involved in collagen biosynthesis and plays a fundamental role in the folding, stability, and intracellular transport of procollagen triple helices. This work reports on a photoactivatable derivative of Hsp47 that allows regulation of collagen biosynthesis within mammalian cells using light. Photoactivatable Hsp47 contains a non-natural light-responsive tyrosine (o-nitro benzyl tyrosine (ONBY)) at Tyr383 position of the protein sequence. This mutation renders Hsp47 inactive toward collagen binding. The inactive, photoactivatable protein is easily uptaken by cells within a few minutes of incubation, and accumulated at the endoplasmic reticulum via retrograde KDEL receptor-mediated uptake. Upon light exposure, the photoactivatable Hsp47 turns into functional Hsp47 in situ. The increased intracellular concentration of Hsp47 results in stimulated secretion of collagen. The ability to promote collagen synthesis on demand, with spatiotemporal resolution, and in diseased state cells is demonstrated in vitro. It is envisioned that photoactivatable Hsp47 allows unprecedented fundamental studies of collagen biosynthesis, matrix biology, and inspires new therapeutic concepts in biomedicine and tissue regeneration.
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Engineering novel biomaterials that mimic closer in vivo scenarios requires the simple and quantitative incorporation of multiple instructive signals to gain a higher level of control and complexity at the cell-matrix interface. Poly(acrylamide) (PAAm) gels are very popular among biology labs as 2D model substrates with defined biochemical and mechanical properties. These gels are cost effective, easy to prepare, reproducible, and available in a wide range of stiffness. However, their functionalization with bioactive ligands (cell adhesive proteins or peptides, growth factors, etc.) in a controlled and functional fashion is not trivial; therefore reproducible and trustable protocols are needed. Amine or thiol groups are ubiquitous in natural or synthetic peptides, proteins, and dyes, and hence routinely used as handles for their immobilization on biomaterials.We describe here the preparation of mechanically defined (0.5-100 kPa, range that approximates the stiffness of most tissues in nature), thin PAAm-based hydrogels supported on a glass substrate and covalently functionalized with amine- or thiol-containing bioligands via simple, robust, and effective protocols.
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Resinas Acrílicas/química , Materiales Biocompatibles/química , Aminas/química , Ligandos , Acoplamiento Oxidativo , Compuestos de Sulfhidrilo/químicaRESUMEN
The ability to guide the growth of neurites is relevant for reconstructing neural networks and for nerve tissue regeneration. Here, a biofunctional hydrogel that allows light-based directional control of axon growth in situ is presented. The gel is covalently modified with a photoactivatable derivative of the short laminin peptidomimetic IKVAV. This adhesive peptide contains the photoremovable group 2-(4'-amino-4-nitro-[1,1'-biphenyl]-3-yl)propan-1-ol (HANBP) on the Lys rest that inhibits its activity. The modified peptide is highly soluble in water and can be simply conjugated to -COOH containing hydrogels via its terminal -NH2 group. Light exposure allows presentation of the IKVAV adhesive motif on a soft hydrogel at desired concentration and at defined position and time point. The photoactivated gel supports neurite outgrowth in embryonic neural progenitor cells culture and allows site-selective guidance of neurites extension. In situ exposure of cell cultures using a scanning laser allows outgrowth of neurites in desired pathways.