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Epithelial-to-mesenchymal transition (EMT) is a key process where cells lose their adhesion properties and augment their invasive properties. α-Actinin4 (ACTN4) is an actin crosslinking protein that responds to mechanical stimuli and is found to be elevated in breast cancer patients. While ACTN4 has been implicated in regulating cancer invasiveness by modulating cytoskeletal organization, its nuclear functions remain much less explored. Here we address this question by first establishing a correlation between nuclear localization and invasiveness in breast cancer cells. Using cancer databases, we then establish a correlation between ACTN4 expression and EMT in breast cancer. Interestingly, TGFß-induced EMT induction in MCF10A normal mammary epithelial cells leads to increased ACTN4 expression and nuclear enrichment. We then show that ACTN4 knockdown in MDA-MB-231 breast cancer cells, which harbor sizeable fraction of nuclear ACTN4, leads to reduced invasiveness and loss of mesenchymal traits. Similar behavior was observed in knockdown cells expressing K255E ACTN4, which is primarily localized to the cytosol. Together, our findings establish a role for nuclear ACTN4 in regulating invasiveness via modulation of EMT.
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Gelatin-based hydrogels have been widely used for wound healing applications. However, increase in ligand density and reduction in pore size with increasing gelatin concentration may delay wound healing by limiting cell infiltration. In this study, we address this shortcoming by combining gelatin with gellan-which is super hydrophilic and non-adhesive to cells. We show that UV crosslinked hybrid gels composed of methacrylated gelatin (GelMA) and methacrylated gellan gum (mGG), possess considerably larger pores and improved mechanical properties compared to GelMA gels. Reduced spreading and reduced formation of focal adhesions on hybrid gels combined with lower contractility and faster detachment upon trypsin-induced de-adhesion suggests that hybrid gels are less adhesive than GelMA gels. Gradual release of fibroblast growth factor (FGF) and silver nanoparticles (AgNPs) incorporated in hybrid gels not only boosts cell migration, but also confers anti-bacterial activity against gram-positive and gram-negative bacteria at concentrations nontoxic to cells. Full thickness wound healing in Wistar rats revealed increased granulation tissue formation in hybrid gels, fastest epithelialization and highest collagen deposition in rats treated with FGF entrapped hybrid gels. Together, our results demonstrate how adhesive tuning and incorporation of bioactive factors can be synergistically combined for achieving complete wound healing.
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Gelatina , Nanopartículas Metálicas , Ratos , Animais , Gelatina/farmacologia , Antibacterianos/farmacologia , Adesivos/farmacologia , Ratos Wistar , Bactérias Gram-Negativas , Bactérias Gram-Positivas , Prata/farmacologia , Cicatrização , Hidrogéis/farmacologiaRESUMO
Cellular heterogeneity and extracellular matrix (ECM) stiffening have been shown to be drivers of breast cancer invasiveness. Here, we examine how stiffness-dependent crosstalk between cancer cells and mesenchymal stem cells (MSCs) within an evolving tumor microenvironment regulates cancer invasion. By analyzing previously published single-cell RNA sequencing datasets, we establish the existence of a subpopulation of cells in primary tumors, secondary sites and circulatory tumor cell clusters of highly aggressive triple-negative breast cancer (TNBC) that co-express MSC and cancer-associated fibroblast (CAF) markers. By using hydrogels with stiffnesses of 0.5, 2 and 5â kPa to mimic different stages of ECM stiffening, we show that conditioned medium from MDA-MB-231 TNBC cells cultured on 2â kPa gels, which mimic the pre-metastatic stroma, drives efficient MSC chemotaxis and induces stable differentiation of MSC-derived CAFs in a TGFß (TGFB1)- and contractility-dependent manner. In addition to enhancing cancer cell proliferation, MSC-derived CAFs on 2â kPa gels maximally boost local invasion and confer resistance to flow-induced shear stresses. Collectively, our results suggest that homing of MSCs at the pre-metastatic stage and their differentiation into CAFs actively drives breast cancer invasion and metastasis in TNBC.
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Neoplasias da Mama , Fibroblastos Associados a Câncer , Células-Tronco Mesenquimais , Neoplasias de Mama Triplo Negativas , Humanos , Feminino , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Diferenciação Celular , Géis , Microambiente Tumoral/genética , Linhagem Celular TumoralRESUMO
Mimivirus and Marseillevirus infections of Acanthamoeba castellanii, like most other viral infections, induce cytopathic effects (CPE). The details of how they bring about CPE and to what extent and how they modify the host cytoskeletal network are unclear. In this study, we compared the rearrangement of the host cytoskeletal network induced by Mimivirus and Marseillevirus upon infection. We show that while both Mimivirus and Marseillevirus infections of A. castellanii cells cause retraction of acanthopodia and depolymerization of the host actin filament network, the Mimivirus infection also results in characteristic cleavage of the host tubulin, a phenomenon not previously reported with any intracellular pathogens. Furthermore, we show that the amoebal tubulin cleavage during Mimivirus infection is a post-replicative event. Because time-lapse microscopy showed that Mimivirus infection leads to the bursting of cells, releasing the virus, we hypothesize that tubulin cleavage together with actin depolymerization during the later stages of Mimivirus assembly is essential for cell lysis due to apoptotic/necrotic cell death. We also characterize the Mimivirus-encoded gp560, a Zn metalloprotease, however, the purified gp560 protein was unable to cleave the commercially available porcine brain tubulin. While protein synthesis is essential for causing the morphological changes in the case of Mimivirus, the proteins which are packaged in the viral capsid along with the genome are sufficient to induce CPE in the case of Marseillevirus. IMPORTANCE In general, intracellular pathogens target the cytoskeletal network to enable their life cycle inside the host. Pathogen-induced changes in the host cell morphology usually accompany global changes in the cytoskeleton resulting in cytopathic effects. While viruses have been shown to use the host actin cytoskeleton for entry and transport during early infection, the role of microtubules in the viral life cycle is only beginning to emerge. Here, we show that the giant viruses Mimivirus and Marseillevirus both induce depolymerization of the actin filament, Mimivirus also causes a characteristic cleavage of tubulin not previously reported for any intracellular pathogen. Because tubulin cleavage occurs late during infection, we hypothesize that tubulin cleavage aids in cell death and lysis rather than establishing infection. The different strategies used by viruses with similar host niches may help them survive in competition.
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Acanthamoeba castellanii , Amoeba , Vírus Gigantes , Mimiviridae , Animais , Suínos , Mimiviridae/genética , Tubulina (Proteína)/metabolismoRESUMO
Fabrication of nanoscale DNA devices to generate 3D nano-objects with precise control of shape, size, and presentation of ligands has shown tremendous potential for therapeutic applications. The interactions between the cell membrane and different topologies of 3D DNA nanostructures are crucial for designing efficient tools for interfacing DNA devices with biological systems. The practical applications of these DNA nanocages are still limited in cellular and biological systems owing to the limited understanding of their interaction with the cell membrane and endocytic pathway. The correlation between the geometry of DNA nanostructures and their internalization efficiency remains elusive. We investigated the influence of the shape and size of 3D DNA nanostructures on their cellular internalization efficiency. We found that one particular geometry, i.e., the tetrahedral shape, is more favored over other designed geometries for their cellular uptake in 2D and 3D cell models. This is also replicable for cellular processes like cell invasion assays in a 3D spheroid model, and passing the epithelial barriers in in vivo zebrafish model systems. Our work provides detailed information for the rational design of DNA nanodevices for their upcoming biological and biomedical applications.
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Nanoestruturas , Peixe-Zebra , Animais , Nanoestruturas/química , DNA/química , Membrana Celular , EndocitoseRESUMO
Stem cell differentiation is dictated by the dynamic crosstalk between cells and their underlying extracellular matrix. While the importance of matrix degradation mediated by enzymes such as matrix metalloproteinases (MMPs) in the context of cancer invasion is well established, the role of MMPs in stem cell differentiation remains relatively unexplored. Here we address this question by assaying MMP expression and activity during differentiation of mouse embryonic stem cells (mESCs) on mouse embryonic fibroblast (MEF) derived matrices (MEFDMs) of varying stiffness and composition. We show that mESC differentiation into different germ layers is associated with expression of several MMPs including MMP-11, 2, 17, 25 and 9, with MMP-9 detected in cell secreted media. Different extents of softening of the different MEFDMs led to altered integrin expression, activated distinct mechanotransduction and metabolic pathways, and induced expression of germ layer-specific markers. Inhibition of MMP proteolytic activity by the broad spectrum MMP inhibitor GM6001 led to alterations in germ layer commitment of the differentiating mESCs. Together, our results illustrate the effect of MMPs in regulating mESC differentiation on engineered cell derived matrices and establish MEFDMs as suitable substrates for understanding molecular mechanisms regulating stem cell development and for regenerative medicine applications.
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Mecanotransdução Celular , Células-Tronco Embrionárias Murinas , Animais , Diferenciação Celular/fisiologia , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Metaloproteinases da Matriz/metabolismo , CamundongosRESUMO
Breast cancer progression features ECM stiffening due to excess deposition and crosslinking of collagen, which dramatically influence tumor behaviour and fate. The mechanisms by which extracellular matrix (ECM) stiffening drives breast cancer invasion is an area of active research. Here we demonstrate the role of exosomes in ECM stiffness triggered breast cancer invasiveness. Using stiffness tuneable hydrogel ECM scaffolds, we show that stiff ECMs promote exosome secretion in a YAP/TAZ pathway-dependent manner. Interestingly, blocking exosome synthesis and secretion by GW4869 abrogated stiffness regulated motility and contractility in breast cancer cells. Reciprocally, exogenous addition of ECM stiffness-tuned exosomes orchestrated a series of changes in cell morphology, adhesion, protrusion dynamics resulting in fostered cell motility and invasion. Proteomic analysis of exosomal lysates followed by overrepresentation analysis and interactome studies revealed enrichment of cell adhesion and cell migration proteins in exosomes from stiff ECM cultures compared to that of soft ones. Quantitative proteomics of exosomes combined with genomic analysis of human breast tumor tissues (TCGA database) identified thrombospondin-1 (THBS1) as a prospective regulator of stiffness-dependent cancer invasion. Knockdown studies confirmed that the pro-invasive effects of stiffness-tuned exosomes are fuelled by exosomal THBS1. We further demonstrated that exosomal THBS1 mediates these stiffness-induced effects by engaging matrix metalloproteinase and focal adhesion kinase. Our studies establish the pivotal role of exosomal communication in ECM stiffness dependent cell migration with exosomal THBS1 as a master regulator of cancer invasion, which can be further exploited as a potential theranostic for improved breast cancer management.
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Neoplasias da Mama , Exossomos , Linhagem Celular Tumoral , Movimento Celular , Matriz Extracelular , Feminino , Humanos , Estudos Prospectivos , ProteômicaRESUMO
The mechanisms by which the mechanoresponsive actin crosslinking protein α-actinin-4 (ACTN4) regulates cell motility and invasiveness remain incompletely understood. Here, we show that, in addition to regulating protrusion dynamics and focal adhesion formation, ACTN4 transcriptionally regulates expression of non-muscle myosin IIB (NMM IIB; heavy chain encoded by MYH10), which is essential for mediating nuclear translocation during 3D invasion. We further show that an indirect association between ACTN4 and NMM IIA (heavy chain encoded by MYH9) mediated by a functional F-actin cytoskeleton is essential for retention of NMM IIA at the cell periphery and modulation of focal adhesion dynamics. A protrusion-dependent model of confined migration recapitulating experimental observations predicts a dependence of protrusion forces on the degree of confinement and on the ratio of nucleus to matrix stiffness. Together, our results suggest that ACTN4 is a master regulator of cancer invasion that regulates invasiveness by controlling NMM IIB expression and NMM IIA localization. This article has an associated First Person interview with the first author of the paper.
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Miosina não Muscular Tipo IIA , Actinina/genética , Actinas/genética , Movimento Celular/genética , Humanos , Cadeias Pesadas de Miosina , Miosina não Muscular Tipo IIA/genética , Miosina não Muscular Tipo IIB/genéticaRESUMO
In comparison to synthetic hydrogels where ligand density and stiffness can be independently tuned, cell responses are expected to deviate on native biopolymer networks where ligand density and stiffness are coupled. Here we probe the tensional homeostasis of fibroblasts on methacrylated gelatin (GelMA) gels, which are widely used in tissue engineering applications. On 5%-15% GelMA gels which are very soft (10-100's of Pa's in stiffness), fibroblasts were found to spread extensively and assemble prominent stress fibers and focal adhesions. Probing of contractile mechanics using trypsin-induced detachment revealed adhesive drag, but not contractility, was sensitive to GelMA concentration. Contractility-altering drugs blebbistatin and nocodazole, which exhibited opposite effects on focal adhesion size, both led to reduction in adhesive drag and cell rounding. However, cell motility was impacted only in nocodazole-treated cells. Collectively, our experiments suggest that on soft GelMA gels, contractility-independent adhesion clustering mediated by high ligand density can drive cell spreading and motility.
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Materiais Biocompatíveis , Adesão Celular/efeitos dos fármacos , Técnicas de Cultura de Células/métodos , Gelatina , Metacrilatos , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Movimento Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Adesões Focais/efeitos dos fármacos , Gelatina/química , Gelatina/farmacologia , Hidrogéis , Ligantes , Metacrilatos/química , Metacrilatos/farmacologia , Camundongos , Células NIH 3T3 , Engenharia TecidualRESUMO
In vivo cell migration is influenced by soluble factors as well as stiffness. Current in vitro strategies mostly account for one of these two factors to study cell migration. To understand the combinatorial effect of stiffness and chemokines on cell behavior, we have developed a microfluidic model to study stiffness-dependent chemotaxis of mesenchymal stem cells (hMSCs). A detailed description of our methodology will help researchers develop microfluidic models that combine these two factors influencing cell behavior. For complete details on the use and execution of this protocol, please refer to Saxena et al. (2018).
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Movimento Celular/fisiologia , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/instrumentação , Quimiocinas , Quimiotaxia , Sinais (Psicologia) , Humanos , Dispositivos Lab-On-A-Chip , Células-Tronco Mesenquimais , Técnicas Analíticas Microfluídicas/instrumentaçãoRESUMO
Quantification of nuclear stiffness is challenging for cells encapsulated within a 3D extracellular matrix (ECM). Here, we describe an experimental setup for measuring microenvironment-dependent tuning of nuclear stiffness using an atomic force microscope (AFM). In our setup, ECM-coated polyacrylamide hydrogels mimic the stiffness of the microenvironment, enabling the measurement of nuclear stiffness using an AFM probe in live cancer cells. For complete details on the use and execution of this protocol, please refer to Das et al. (2019) (https://doi.org/10.1016/j.matbio.2019.01.001).
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Núcleo Celular , Matriz Extracelular , Microscopia de Força Atômica , Neoplasias , Microambiente Tumoral , Resinas Acrílicas , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Humanos , Neoplasias/metabolismo , Neoplasias/ultraestruturaRESUMO
Phenotypic heterogeneity is increasingly acknowledged to confer several advantages to cancer progression and drug resistance. Here, we probe the collective importance of heterogeneity in cell size and deformability in breast cancer invasion. A computational model of invasion of a heterogeneous cell aggregate predicts that combined heterogeneity in cell size and deformability enhances invasiveness of the whole population, with maximum invasiveness at intermediate cell-cell adhesion. We then show that small cells of varying deformability, a subpopulation predicted to be enriched at the invasive front, exhibit considerable overlap with the biophysical properties of cancer stem cells (CSCs). In MDA-MB-231 cells, these include CD44 hi CD24- mesenchymal CSCs, which are small and soft, and CD44 hi CD24+ hybrid CSCs, which exhibit a wide range of size and deformability. We validate our predictions by tracking the pattern of cell invasion from spheroids implanted in three-dimensional collagen gels, wherein we show temporal enrichment of CD44 hi cells at the invasive front. Collectively, our results illustrate the advantages imparted by biophysical heterogeneity in enhancing cancer invasiveness.This article has an associated First Person interview with the first author of the paper.
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Neoplasias da Mama , Antígeno CD24 , Neoplasias da Mama/genética , Adesão Celular , Linhagem Celular Tumoral , Tamanho Celular , Feminino , Humanos , Receptores de Hialuronatos , Invasividade Neoplásica , Células-Tronco NeoplásicasRESUMO
Impaired wound healing represents one of the most debilitating side effects of Diabetes mellitus. Though the role of fibroblasts in wound healing is well-known, the extent to which their function is altered in the context of diabetes remains incompletely understood. Here, we address this question by comparing the phenotypes of healthy dermal fibroblasts (HDFs) and diabetic dermal fibroblasts (DDFs). We show that DDFs are more elongated but less motile and less contractile than HDFs. Reduced motility of DDFs is attributed to formation of larger focal adhesions stabilized by a bulky glycocalyx, associated with increased expression of the cell surface glycoprotein mucin 16 (MUC 16). Disruption of the glycocalyx not only restored DDF motility to levels comparable to that of HDFs, but also led to increased proliferation and collagen synthesis. Collectively, our results illustrate the influence of glycocalyx disruption on mechanics of diabetic fibroblasts relevant to cell motility.
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Colágeno/metabolismo , Diabetes Mellitus/metabolismo , Fibroblastos/citologia , Glicocálix/metabolismo , Adulto , Antígeno Ca-125/metabolismo , Estudos de Casos e Controles , Movimento Celular , Proliferação de Células , Células Cultivadas , Fibroblastos/metabolismo , Adesões Focais/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Regulação para CimaRESUMO
Large nuclear deformations during migration through confined spaces have been associated with nuclear membrane rupture and DNA damage. However, the stresses associated with nuclear damage remain unclear. Here, using a quasi-static plane strain finite element model, we map evolution of nuclear shape and stresses during confined migration of a cell through a deformable matrix. Plastic deformation of the nucleus observed for a cell with stiff nucleus transiting through a stiffer matrix lowered nuclear stresses, but also led to kinking of the nuclear membrane. In line with model predictions, transwell migration experiments with fibrosarcoma cells showed that while nuclear softening increased invasiveness, nuclear stiffening led to plastic deformation and higher levels of DNA damage. In addition to highlighting the advantage of nuclear softening during confined migration, our results suggest that plastic deformations of the nucleus during transit through stiff tissues may lead to bending-induced nuclear membrane disruption and subsequent DNA damage.
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Movimento Celular/fisiologia , Núcleo Celular/fisiologia , Modelos Biológicos , Linhagem Celular Tumoral , Dano ao DNA , Análise de Elementos Finitos , Humanos , Membrana Nuclear/fisiologiaRESUMO
Growth cone-mediated axonal outgrowth and accurate synaptic targeting are central to brain morphogenesis. Translocation of the growth cone necessitates mechanochemical regulation of cell-extracellular matrix interactions and the generation of propulsive traction forces onto the growth environment. However, the molecular mechanisms subserving force generation by growth cones remain poorly characterized. The formin family member, Fmn2, has been identified earlier as a regulator of growth cone motility. Here, we explore the mechanisms underlying Fmn2 function in the growth cone. Evaluation of multiple components of the adhesion complexes suggests that Fmn2 regulates point contact stability. Analysis of F-actin retrograde flow reveals that Fmn2 functions as a clutch molecule and mediates the coupling of the actin cytoskeleton to the growth substrate, via point contact adhesion complexes. Using traction force microscopy, we show that the Fmn2-mediated clutch function is necessary for the generation of traction stresses by neurons. Our findings suggest that Fmn2, a protein associated with neurodevelopmental and neurodegenerative disorders, is a key regulator of a molecular clutch activity and consequently motility of neuronal growth cones.
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Forminas/genética , Cones de Crescimento , Proteínas Nucleares/genética , Actinas , Movimento Celular , NeurôniosRESUMO
A coumarin-appended calixarene derivative ( CouC4A ) and a hybrid material generated by covalently linking this onto a silica surface ( CouC4A@SiO 2 ) were synthesized and were characterized by various analytical, spectroscopy, and microscopy methods. Both these materials are capable of sensing Fe3+ with greater sensitivity and selectivity. The sensitivity is enhanced by 30,000 fold on going from a simple solution phase to the silica surface with the limit of Fe3+ detection being 1.75 ± 0.4 pM when CouC4A@SiO 2 is used, and the sensing is partially reversible with phosphates, while it is completely reversible with adenosine 5'-triphosphate (ATP). While the calix precursor, CouC4A , has a limitation to work in water, anchoring this onto SiO2 endowed it with the benefit of its use in water as well as in buffer and thereby extends its application toward Fe3+ sensing even in the biorelevant medium such as fetal bovine serum and human serum. The hybrid material is biocompatible and shows â¼90% cell viability in the case of MDA-MB231 and 3T3 cell lines. CouC4A@SiO 2 functions as a reversible sensor for Fe3+ with the use of ATP in vitro as well as in biological cells. Thus, the inorganic-organic hybrid material, such as, CouC4A@SiO 2 , is an indispensable material for sensitive and selective detection of Fe3+ in a picomolar range in solution and in nanomolar to micromolar range in biorelevant fluids and biological cells, respectively.
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Substantial number of breast cancer (BC) patients undergoing radiation therapy (RT) develop local recurrence over time. During RT therapy, cells can gradually acquire resistance implying adaptive radioresistance. Here we probe the mechanisms underlying this acquired resistance by first establishing radioresistant lines using ZR-75-1 and MCF-7 BC cells through repeated exposure to sub-lethal fractionated dose of 2Gy up to 15 fractions. Radioresistance was found to be associated with increased cancer stem cells (CSCs), and elevated EpCAM expression in the cell population. A retrospective analysis of TCGA dataset indicated positive correlation of high EpCAM expression with poor response to RT. Intriguingly, elevated EpCAM expression in the radioresistant CSCs raise the bigger question of how this biomarker expression contributes during radiation treatment in BC. Thereafter, we establish EpCAM overexpressing ZR-75-1 cells (ZR-75-1EpCAM), which conferred radioresistance, increased stemness through enhanced AKT activation and induced a hybrid epithelial/mesenchymal phenotype with enhanced contractility and invasiveness. In line with these observations, orthotopic implantation of ZR-75-1EpCAM cells exhibited faster growth, lesser sensitivity to radiation therapy and increased lung metastasis than baseline ZR-75-1 cells in mice. In summary, this study shows that similar to radioresistant BC cells, EpCAM overexpressing cells show high degree of plasticity and heterogeneity which ultimately induces radioresistant and metastatic behavior of cancer cells, thus aggravating the disease condition.
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During amoeboidal migration, cancer cells migrate in a protease-independent manner by squeezing through pre-existing gaps in the extracellular matrix (ECM). However, the extent to which cells alter their physical properties in order to sustain this mode of migration remains unclear. Here, we address this question by documenting biophysical changes in the properties of highly invasive MDA-MB-231 and HT-1080 cells upon inhibition of pericellular proteolysis. Remarkably, treatment with the broad spectrum MMP inhibitor GM6001 not only induces cell rounding and loss of actomyosin contractility, but also induces nuclear softening via increased phosphorylation of the nuclear membrane protein lamin A/C. Though nuclear softening is necessary for sustaining migration through sub-nuclear sized transwell pores, it is not sufficient. In addition, baseline levels of contractility mediating pore entry and peri-nuclear actin inside the pores mediating pore migration are also required. Taken together, our results suggest that protease-independent migration through sub-nuclear sized pre-existing tracks is enabled by deformation of a softened nucleus by contractility and the peri-nuclear actin network.
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Actomiosina/metabolismo , Dipeptídeos/farmacologia , Lamina Tipo A/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Neoplasias/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeo Hidrolases/metabolismo , Fosforilação , Proteólise/efeitos dos fármacosRESUMO
Gelatin-based hydrogels have received particular attention for tissue-engineering applications given their biocompatibility, ease of tuning their physical properties through chemical modifications, and incorporation of antibacterial activity. While several studies have focused on the detailed quantification of biomechanical properties of these gels, considerably less attention has been paid to understanding how adhesivity of these gels impacts single as well as collective cell migration, which directly determines the efficacy of wound healing. In this study, we address this question by quantifying fibroblast motility and antibacterial activity of silver nanoparticle (AgNP)-entrapped methacrylated gelatin (GelMA) hydrogels. Using 5 and 15% GelMA soft gels cross-linked with 1 min UV exposure, we first show that cells spread more and migrate faster on 15% GelMA gels. Next, we show that â¼10 nm AgNPs entrapped in 15% GelMA gels get released over a time-scale greater than 72 h and exhibit antibacterial activity against both Gram-positive and Gram-negative bacteria at concentrations nontoxic to cells. Finally, using a polydimethylsiloxane (PDMS) device for simulating wound healing, we show that closure of â¼800 µm gaps on GelMA gels is significantly faster compared with other conditions. Together, our findings illustrate the potential of AgNP-entrapped soft GelMA gels as scaffolds for achieving accelerated wound healing of deep dermal wounds by enabling fast fibroblast migration and minimization of microbial infections.
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Differentiation of stem cells into neurogenic lineage is of great interest for treatment of neurodegenerative diseases. While the role of chemical cues in regulating stem cell fate is well appreciated, the identification of physical cues has revolutionized the field of tissue engineering leading to development of scaffolds encoding one or more physical cues for inducing stem cell differentiation. In this study, using human mesenchymal stem cells (hMSCs) and mouse embryonic stem cells (mESCs), we have tested if stiffness and topography can be collectively tuned for inducing neuronal differentiation by culturing these cells on polyacrylamide hydrogels of varying stiffness (5, 10, and 20 kPa) containing rectangular grooves (10, 15, and 25 µm in width). While hMSCs maximally elongate and express neuronal markers on soft 5 kPa gels containing 10/15 µm grooves, single mESCs are unable to sense topographical features when cultured directly on grooved gels. However, this inability to sense topography is rescued by priming mESCs initially on soft 1 kPa flat gels and then replating these cells onto the grooved gels. Compared to direct culture on the grooved gels, this sequential adaptation increases both viability as well as neuronal differentiation. However, this two-step process does not enhance neuronal marker expression in hMSCs. In addition to highlighting important differences between hMSCs and mESCs in their responsiveness to physical cues, our study suggests that conditioning on soft substrates is essential for inducing topography-mediated neuronal differentiation in mESCs.