RESUMO
Boron nitride nanotubes (BNNTs) have unique physical properties, of value in biomedical applications; however, their dispersion and functionalization represent a critical challenge in their successful employment as biomaterials. In the present study, we report a process for the efficient disentanglement of BNNTs via a dual surfactant/polydopamine (PD) process. High-resolution transmission electron microscopy (HR-TEM) indicated that individual BNNTs become coated with a uniform PD nanocoating, which significantly enhanced dispersion of BNNTs in aqueous solutions. Furthermore, the cytocompatibility of PD-coated BNNTs was assessed in vitro with cultured human osteoblasts (HOBs) at concentrations of 1, 10, and 30 µg/mL and over three time-points (24, 48, and 72 h). In this study it was demonstrated that PD-functionalized BNNTs become individually localized within the cytoplasm by endosomal escape and that concentrations of up to 30 µg/mL of PD-BNNTs were cytocompatible in HOBs cells following 72 h of exposure.
Assuntos
Materiais Biocompatíveis/farmacologia , Compostos de Boro/química , Indóis/química , Nanotubos/química , Polímeros/química , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacocinética , Compostos de Boro/farmacocinética , Soluções Tampão , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Humanos , Indóis/farmacocinética , Microscopia Eletrônica de Transmissão , Osteoblastos/efeitos dos fármacos , Espectroscopia Fotoeletrônica , Polímeros/farmacocinética , Espectrometria por Raios XRESUMO
Focal adhesions (FAs) are nanoscale complexes containing clustered integrin receptors and intracellular structural and signaling proteins that function as principal sites of mechanotransduction in part via promoting the nuclear translocation and activation of the transcriptional coactivator yes-associated protein (YAP). Knockdown of FA proteins such as focal adhesion kinase (FAK), talin, and vinculin can prevent YAP nuclear localization. However, the mechanism(s) of action remain poorly understood. Herein, we investigated the role of different functional domains in vinculin, talin, and FAK in regulating YAP nuclear localization. Using genetic or pharmacological inhibition of fibroblasts and human mesenchymal stem cells (hMSCs) adhering to deformable substrates, we find that disruption of vinculin-talin binding versus talin-FAK binding reduces YAP nuclear localization and transcriptional activity via different mechanisms. Disruption of vinculin-talin binding or knockdown of talin-1 reduces nuclear size, traction forces, and YAP nuclear localization. In contrast, disruption of the talin binding site on FAK or elimination of FAK catalytic activity did not alter nuclear size yet still prevented YAP nuclear localization and activity. These data support both nuclear tension-dependent and independent models for matrix stiffness-regulated YAP nuclear localization. Our results highlight the importance of vinculin-talin-FAK interactions at FAs of adherent cells, controlling YAP nuclear localization and activity.
Assuntos
Núcleo Celular , Mecanotransdução Celular , Talina , Vinculina , Proteínas de Sinalização YAP , Talina/metabolismo , Vinculina/metabolismo , Humanos , Núcleo Celular/metabolismo , Proteínas de Sinalização YAP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fatores de Transcrição/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Animais , Adesões Focais/metabolismo , Camundongos , Fibroblastos/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Ligação ProteicaRESUMO
Cell-extracellular matrix forces provide pivotal signals regulating diverse physiological and pathological processes. Although mechanobiology has been widely studied in two-dimensional configurations, limited research has been conducted in three-dimensional (3D) systems due to the complex nature of mechanics and cellular behaviors. In this study, we established a platform integrating a well-defined synthetic hydrogel system (PEG-4MAL) with 3D traction force microscopy (TFM) methodologies to evaluate deformation and force responses within synthetic microenvironments, providing insights that are not tractable using biological matrices because of the interdependence of biochemical and biophysical properties and complex mechanics. We dissected the contributions of adhesive peptide density and polymer density, which determines hydrogel stiffness, to 3D force generation for fibroblasts. A critical threshold of adhesive peptide density at a constant matrix elasticity is required for cells to generate 3D forces. Furthermore, matrix displacements and strains decreased with matrix stiffness whereas stresses, and tractions increased with matrix stiffness until reaching constant values at higher stiffness values. Finally, Rho-kinase-dependent contractility and vinculin expression are required to generate significant 3D forces in both collagen and synthetic hydrogels. This research establishes a tunable platform for the study of mechanobiology and provides new insights into how cells sense and transmit forces in 3D.
Assuntos
Hidrogéis , Tração , Adesivos , Matriz Extracelular/química , Hidrogéis/química , Peptídeos/análise , PolímerosRESUMO
Understanding the immune response to hydrogel implantation is critical for the design of immunomodulatory biomaterials. To study the progression of inflammation around poly(ethylene glycol) hydrogels presenting Arg-Gly-Asp (RGD) peptides and vascular endothelial growth factor, we used temporal analysis of high-dimensional flow cytometry data paired with intravital imaging, immunohistochemistry, and multiplexed proteomic profiling. RGD-presenting hydrogels created a reparative microenvironment promoting CD206+ cellular infiltration and revascularization in wounded dorsal skin tissue. Unbiased clustering algorithms (SPADE) revealed significant phenotypic transition shifts as a function of the cell-adhesion hydrogel properties. SPADE identified an intermediate macrophage subset functionally regulating in vivo cytokine secretion that was preferentially recruited for RGD-presenting hydrogels, whereas dendritic cell subsets were preferentially recruited to RDG-presenting hydrogels. Last, RGD-presenting hydrogels controlled macrophage functional cytokine secretion to direct polarization and vascularization. Our studies show that unbiased clustering of single-cell data provides unbiased insights into the underlying immune response to engineered materials.
Assuntos
Hidrogéis , Fator A de Crescimento do Endotélio Vascular , Materiais Biocompatíveis/química , Análise por Conglomerados , Citocinas , Hidrogéis/química , Imunidade , Oligopeptídeos/química , ProteômicaRESUMO
How adhesive forces are transduced and integrated into biochemical signals at focal adhesions (FAs) is poorly understood. Using cells adhering to deformable micropillar arrays, we demonstrate that traction force and FAK localization as well as traction force and Y397-FAK phosphorylation are linearly coupled at individual FAs on stiff, but not soft, substrates. Similarly, FAK phosphorylation increases linearly with external forces applied to FAs using magnetic beads. This mechanosignaling coupling requires actomyosin contractility, talin-FAK binding, and full-length vinculin that binds talin and actin. Using an in vitro 3D biomimetic wound healing model, we show that force-FAK signaling coupling coordinates cell migration and tissue-scale forces to promote microtissue repair. A simple kinetic binding model of talin-FAK interactions under force can recapitulate the experimental observations. This study provides insights on how talin and vinculin convert forces into FAK signaling events regulating cell migration and tissue repair.
Assuntos
Quinase 1 de Adesão Focal/metabolismo , Adesões Focais/metabolismo , Modelos Biológicos , Actomiosina/metabolismo , Animais , Fenômenos Biomecânicos , Biomimética , Movimento Celular/fisiologia , Células Cultivadas , Fibroblastos/metabolismo , Quinase 1 de Adesão Focal/deficiência , Quinase 1 de Adesão Focal/genética , Mecanotransdução Celular , Camundongos , Camundongos Knockout , Fosforilação , RNA Interferente Pequeno/genética , Transdução de Sinais , Talina/antagonistas & inibidores , Talina/genética , Talina/metabolismo , Cicatrização/fisiologiaRESUMO
Tendon disease constitutes an unmet clinical need and remains a critical challenge in the field of orthopaedic surgery. Innovative solutions are required to overcome the limitations of current tendon grafting approaches, and bioelectronic therapies show promise in treating musculoskeletal diseases, accelerating functional recovery through the activation of tissue regeneration-specific signaling pathways. Self-powered bioelectronic devices, particularly piezoelectric materials, represent a paradigm shift in biomedicine, negating the need for battery or external powering and complementing existing mechanotherapy to accelerate the repair processes. Here, the dynamic response of tendon cells to a piezoelectric collagen-analogue scaffold comprised of aligned nanoscale fibers made of the ferroelectric material poly(vinylidene fluoride-co-trifluoroethylene) is shown. It is demonstrated that motion-powered electromechanical stimulation of tendon tissue through piezo-bioelectric device results in ion channel modulation in vitro and regulates specific tissue regeneration signaling pathways. Finally, the potential of the piezo-bioelectronic device in modulating the progression of tendinopathy-associated processes in vivo, using a rat Achilles acute injury model is shown. This study indicates that electromechanical stimulation regulates mechanosensitive ion channel sensitivity and promotes tendon-specific over non-tenogenic tissue repair processes.
Assuntos
Eletrônica , Canais Iônicos/metabolismo , Tendões/fisiologia , Engenharia Tecidual/métodos , Animais , Colágeno/química , Módulo de Elasticidade , Estimulação Elétrica , Hidrocarbonetos Fluorados/química , Ratos , Regeneração/fisiologia , Transdução de Sinais , Tendões/citologia , Engenharia Tecidual/instrumentação , Alicerces Teciduais/química , Compostos de Vinila/químicaRESUMO
Translation of transplanted alginate-encapsulated pancreatic islets to treat type 1 diabetes has been hindered by inconsistent long-term efficacy. This loss of graft function can be partially attributed to islet dysfunction associated with the destruction of extracellular matrix (ECM) interactions during the islet isolation process as well as immunosuppression-associated side effects. This study aims at recapitulating islet-ECM interactions by the direct functionalization of alginate with the ECM-derived peptides RGD, LRE, YIGSR, PDGEA, and PDSGR. Peptide functionalization is controlled in a concentration-dependent manner and its presentation is found to be homogeneous across the microcapsule environment. Preweaned porcine islets are encapsulated in peptide-functionalized alginate microcapsules, and those encapsulated in RGD-functionalized alginate displays enhanced viability and glucose-stimulated insulin release. Effects are RGD-specific and not observed with its scrambled control RDG nor with LRE, YIGSR, PDGEA, and PDSGR. This study supports the sustained presentation of ECM-derived peptides in helping to maintain health of encapsulated pancreatic islets and may aid in prolonging longevity of encapsulated islet grafts.
Assuntos
Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Alginatos , Animais , Matriz Extracelular , Insulina , Peptídeos/farmacologia , SuínosRESUMO
There is a pressing clinical need to develop cell-based bone therapies due to a lack of viable, autologous bone grafts and a growing demand for bone grafts in musculoskeletal surgery. Such therapies can be tissue engineered and cellular, such as osteoblasts, combined with a material scaffold. Because mesenchymal stem cells (MSCs) are both available and fast growing compared to mature osteoblasts, therapies that utilize these progenitor cells are particularly promising. We have developed a nanovibrational bioreactor that can convert MSCs into bone-forming osteoblasts in two- and three-dimensional, but the mechanisms involved in this osteoinduction process remain unclear. Here, to elucidate this mechanism, we use increasing vibrational amplitude, from 30 nm (N30) to 90 nm (N90) amplitudes at 1000 Hz and assess MSC metabolite, gene, and protein changes. These approaches reveal that dose-dependent changes occur in MSCs' responses to increased vibrational amplitude, particularly in adhesion and mechanosensitive ion channel expression and that energetic metabolic pathways are activated, leading to low-level reactive oxygen species (ROS) production and to low-level inflammation as well as to ROS- and inflammation-balancing pathways. These events are analogous to those that occur in the natural bone-healing processes. We have also developed a tissue engineered MSC-laden scaffold designed using cells' mechanical memory, driven by the stronger N90 stimulation. These mechanistic insights and cell-scaffold design are underpinned by a process that is free of inductive chemicals.
Assuntos
Células-Tronco Mesenquimais , Diferenciação Celular , Humanos , Inflamação , Osteogênese , Espécies Reativas de Oxigênio , Engenharia Tecidual , Alicerces TeciduaisRESUMO
Polymeric capsules exhibit significant potential for therapeutic applications as microreactors, where the bio-chemical reactions of interest are efficiently performed in a spatial and time defined manner due to the encapsulation of an active biomolecule (e.g., enzyme) and control over the transfer of reagents and products through the capsular membrane. In this work, catalase loaded polymer capsules functionalized with an external layer of tannic acid (TA) are fabricated via a layer-by-layer approach using calcium carbonate as a sacrificial template. The capsules functionalised with TA exhibit a higher scavenging capacity for hydrogen peroxide and hydroxyl radicals, suggesting that the external layer of TA shows intrinsic antioxidant properties, and represents a valid strategy to increase the overall antioxidant potential of the developed capsules. Additionally, the hydrogen peroxide scavenging capacity of the capsules is enhanced in the presence of the encapsulated catalase. The capsules prevent oxidative stress in an in vitro inflammation model of degenerative disc disease. Moreover, the expression of matrix metalloproteinase-3 (MMP-3), and disintegrin and metalloproteinase with thrombospondin motif-5 (ADAMTS-5), which represents the major proteolytic enzymes in intervertebral disc, are attenuated in the presence of the polymer capsules. This platform technology exhibits potential to reduce oxidative stress, a key modulator in the pathology of a broad range of inflammatory diseases. STATEMENT OF SIGNIFICANCE: Oxidative stress damages important cell structures leading to cellular apoptosis and senescence, for numerous disease pathologies including cancer, neurodegeneration or osteoarthritis. Thus, the development of biomaterials-based systems to control oxidative stress has gained an increasing interest. Herein, polymer capsules loaded with catalase and functionalized with an external layer of tannic acid are fabricated, which can efficiently scavenge important reactive oxygen species (i.e., hydroxyl radicals and hydrogen peroxide) and modulate extracellular matrix activity in an in vitro inflammation model of nucleus pulposus. The present work represents accordingly, an important advance in the development and application of polymer capsules with antioxidant properties for the treatment of oxidative stress, which is applicable for multiple inflammatory disease targets.
Assuntos
Antioxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Polímeros/farmacologia , Proteína ADAMTS5/metabolismo , Animais , Cápsulas , Catalase/metabolismo , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Peróxido de Hidrogênio/toxicidade , Metaloproteinase 3 da Matriz/metabolismo , Núcleo Pulposo/efeitos dos fármacos , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Taninos/químicaRESUMO
AIM: Medium chain length-polyhydroxyalkanoate/multi-walled carbon nanotube (MWCNTs) nanocomposites with a range of mechanical and electrochemical properties were fabricated via assisted dispersion and solvent casting, and their suitability as neural interface biomaterials was investigated. MATERIALS & METHODS: Mechanical and electrical properties of medium chain length-polyhydroxyalkanoate/MWCNTs nanocomposite films were evaluated by tensile test and electrical impedance spectroscopy, respectively. Primary rat mesencephalic cells were seeded on the composites and quantitative immunostaining of relevant neural biomarkers, and electrical stimulation studies were performed. RESULTS: Incorporation of MWCNTs to the polymeric matrix modulated the mechanical and electrical properties of resulting composites, and promoted differential cell viability, morphology and function as a function of MWCNT concentration. CONCLUSION: This study demonstrates the feasibility of a green thermoplastic MWCNTs nanocomposite for potential use in neural interfacing applications.
Assuntos
Elastômeros/química , Nanocompostos/química , Nanotubos de Carbono/química , Neurônios/fisiologia , Poli-Hidroxialcanoatos/química , Animais , Materiais Biocompatíveis , Cálcio/química , Cálcio/metabolismo , Adesão Celular , Técnicas de Cultura de Células , Sobrevivência Celular , Condutividade Elétrica , Estimulação Elétrica , Estudos de Viabilidade , Humanos , Fenômenos Mecânicos , Mesencéfalo/citologia , Tamanho da Partícula , Ratos Sprague-Dawley , Propriedades de Superfície , Engenharia TecidualRESUMO
The development of responsive biomaterials capable of demonstrating modulated function in response to dynamic physiological and mechanical changes in vivo remains an important challenge in bone tissue engineering. To achieve long-term repair and good clinical outcomes, biologically responsive approaches that focus on repair and reconstitution of tissue structure and function through drug release, receptor recognition, environmental responsiveness and tuned biodegradability are required. Traditional orthopedic materials lack biomimicry, and mismatches in tissue morphology, or chemical and mechanical properties ultimately accelerate device failure. Multiple stimuli have been proposed as principal contributors or mediators of cell activity and bone tissue formation, including physical (substrate topography, stiffness, shear stress and electrical forces) and biochemical factors (growth factors, genes or proteins). However, optimal solutions to bone regeneration remain elusive. This review will focus on biological and physicomechanical considerations currently being explored in bone tissue engineering.