RESUMO
The development of multicellular complex organisms relies on coordinated signaling from the microenvironment, including both biochemical and mechanical interactions. To better understand developmental biology, increasingly sophisticated in vitro systems are needed to mimic these complex extracellular features. In this Primer, we explore how engineered hydrogels can serve as in vitro culture platforms to present such signals in a controlled manner and include examples of how they have been used to advance our understanding of developmental biology.
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Hidrogéis , Transdução de SinaisRESUMO
Cells integrate mechanical cues to direct fate specification to maintain tissue function and homeostasis. While disruption of these cues is known to lead to aberrant cell behavior and chronic diseases, such as tendinopathies, the underlying mechanisms by which mechanical signals maintain cell function are not well understood. Here, we show using a model of tendon de-tensioning that loss of tensile cues in vivo acutely changes nuclear morphology, positioning, and expression of catabolic gene programs, resulting in subsequent weakening of the tendon. In vitro studies using paired ATAC/RNAseq demonstrate that the loss of cellular tension rapidly reduces chromatin accessibility in the vicinity of Yap/Taz genomic targets while also increasing expression of genes involved in matrix catabolism. Concordantly, the depletion of Yap/Taz elevates matrix catabolic expression. Conversely, overexpression of Yap results in a reduction of chromatin accessibility at matrix catabolic gene loci, while also reducing transcriptional levels. The overexpression of Yap not only prevents the induction of this broad catabolic program following a loss of cellular tension, but also preserves the underlying chromatin state from force-induced alterations. Taken together, these results provide novel mechanistic details by which mechanoepigenetic signals regulate tendon cell function through a Yap/Taz axis.
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Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteínas de Sinalização YAP , Cromatina/genética , Cromatina/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Homeostase , Transdução de Sinais/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAP/genética , Proteínas de Sinalização YAP/metabolismo , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/genética , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/metabolismoRESUMO
Intervertebral disc degeneration is a leading cause of chronic low back pain. Cell-based strategies that seek to treat disc degeneration by regenerating the central nucleus pulposus (NP) hold significant promise, but key challenges remain. One of these is the inability of therapeutic cells to effectively mimic the performance of native NP cells, which are unique amongst skeletal cell types in that they arise from the embryonic notochord. In this study, we use single cell RNA sequencing to demonstrate emergent heterogeneity amongst notochord-derived NP cells in the postnatal mouse disc. Specifically, we established the existence of progenitor and mature NP cells, corresponding to notochordal and chondrocyte-like cells, respectively. Mature NP cells exhibited significantly higher expression levels of extracellular matrix (ECM) genes including aggrecan, and collagens II and VI, along with elevated transforming growth factor-beta and phosphoinositide 3 kinase-protein kinase B signaling. Additionally, we identified Cd9 as a novel surface marker of mature NP cells, and demonstrated that these cells were localized to the NP periphery, increased in numbers with increasing postnatal age, and co-localized with emerging glycosaminoglycan-rich matrix. Finally, we used a goat model to show that Cd9+ NP cell numbers decrease with moderate severity disc degeneration, suggesting that these cells are associated with maintenance of the healthy NP ECM. Improved understanding of the developmental mechanisms underlying regulation of ECM deposition in the postnatal NP may inform improved regenerative strategies for disc degeneration and associated low back pain.
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Degeneração do Disco Intervertebral , Disco Intervertebral , Dor Lombar , Núcleo Pulposo , Camundongos , Animais , Núcleo Pulposo/metabolismo , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/metabolismo , Disco Intervertebral/metabolismo , Notocorda/metabolismo , Dor Lombar/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Análise de Sequência de RNARESUMO
OBJECTIVES: Fibroses are disorders linked to persistence of myofibroblasts due to biochemical (e.g., Transforming growth factor-ß) and biophysical cues (e.g., a stiff microenvironment). In the context of osteoarthritis, fibrotic changes in the joint-lining synovium have been linked with disease progression. The objective of this study was to probe synovial fibroblast mechanobiology and how essential functions (i.e., lubrication) are altered in fibrotic environments. DESIGN: Both ex vivo and in vitro synovium models were assessed for fibrotic and lubrication biomarkers to better understand the role of mechanobiology and lubrication. Additionally, in vitro, work on small molecules targeting mechanobiology was assessed. RESULTS: Our results indicated that modulating mechanobiology could rescue the fibrotic phenotype instigated by stiffening microenvironment that resulted in altered lubricant expression. A small molecule therapeutic, fasudil, blocked ROCK-mediated contractility and this inhibition of the fibrotic mechano-response of synovial fibroblasts restored proper lubrication function, providing insight into mechanisms of disease progression as well as a new avenue for therapeutic development. CONCLUSION: This study identifies synovial fibrosis as a condition that potentially has joint-wide deficits through inhibiting lubrication. Additionally, modulating mechanobiology (i.e., ROCK-mediated contractility) may pose a potential target for small molecule therapies that can be delivered to the joint space. CLASSIFICATION: Applied Biological Sciences.
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Membrana Sinovial , Humanos , Lubrificação , Fibrose , Membrana Sinovial/metabolismo , Biofísica , Progressão da DoençaRESUMO
Varying degrees of hydroxyapatite (HA) surface functionalization have been implicated as the primary driver of differential osteogenesis observed in infiltrating cells. The ability to reliably create spatially controlled areas of mineralization in composite engineered tissues is of growing interest in the field, and the use of HA-functionalized biomaterials may provide a robust solution to this challenge. In this study, we successfully fabricated polycaprolactone salt-leached scaffolds with two levels of a biomimetic calcium phosphate coating to examine their effects on MSC osteogenesis. Longer duration coating in simulated body fluid (SBF) led to increased HA crystal nucleation within scaffold interiors as well as more robust HA crystal formation on scaffold surfaces. Ultimately, the increased surface stiffness of scaffolds coated in SBF for 7 days in comparison to scaffolds coated in SBF for 1 day led to more robust osteogenesis of MSCs in vitro without the assistance of osteogenic signaling molecules. This study also demonstrated that the use of SBF-based HA coatings can promote higher levels of osteogenesis in vivo. Finally, when incorporated as the endplate region of a larger tissue-engineered intervertebral disc replacement, HA coating did not induce mineralization in or promote cell migration out of neighboring biomaterials. Overall, these results verified tunable biomimetic HA coatings as a promising biomaterial modification to promote discrete regions of mineralization within composite engineered tissues.
Assuntos
Materiais Biocompatíveis , Osseointegração , Engenharia Tecidual/métodos , Osteogênese , Durapatita/química , Alicerces Teciduais/química , Materiais Revestidos Biocompatíveis/farmacologia , Materiais Revestidos Biocompatíveis/químicaRESUMO
The meniscus serves important load-bearing functions and protects the underlying articular cartilage. Unfortunately, meniscus tears are common and impair the ability of the meniscus to distribute loads, increasing the risk of developing osteoarthritis. Therefore, surgical repair of the meniscus is a frequently performed procedure; however, repair does not always prevent osteoarthritis. This is hypothesized to be due to altered joint loading post-injury and repair, where the functional deficit of the meniscus prevents it from performing its role of distributing forces. The objective of this study was to quantify joint kinematics in an intact joint, after a meniscus root tear, and after suture repair in cadaveric porcine knees, a frequently used in vivo model. We utilized an magnetic resonance images-compatible loading device and novel use of a T1 vibe sequence to measure meniscus and femur displacements under physiological axial loads. We found that anterior root tear led to large meniscus displacements under physiological axial loading and that suture anchor repair reduced these displacements but did not fully restore intact joint kinematics. After tear and repair, the anterior region of the meniscus moved posteriorly and medially as it was forced out of the joint space under loading, while the posterior region had small displacements as the posterior attachment acted as a hinge about which the meniscus pivoted in the axial plane. Methods from this study can be applied to assess altered joint kinematics following human knee injuries and evaluate repair strategies aimed to restore joint kinematics.
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Menisco , Osteoartrite , Lesões do Menisco Tibial , Humanos , Suínos , Animais , Meniscos Tibiais/cirurgia , Cadáver , Articulação do Joelho , Fenômenos Biomecânicos , Imageamento por Ressonância MagnéticaRESUMO
Reproduction in procellariiform birds is characterized by a single egg clutch, slow development, a long breeding season and obligate biparental care. Female Leach's Storm Petrels Hydrobates leucorhous, nearly monomorphic members of this order, produce eggs that are between 20 and 25% of adult body weight. We tested whether female foraging behaviour differs from male foraging behaviour during the ~ 44-day incubation period across seven breeding colonies in the Northwest Atlantic. Over six breeding seasons, we used a combination of Global Positioning System and Global Location Sensor devices to measure characteristics of individual foraging trips during the incubation period. Females travelled significantly greater distances and went farther from the breeding colony than did males on individual foraging trips. For both sexes, the longer the foraging trip, the greater the distance. Independent of trip duration, females travelled farther, and spent a greater proportion of their foraging trips prospecting widely as defined by behavioural categories derived from a Hidden Markov Model. For both sexes, trip duration decreased with date. Sex differences in these foraging metrics were apparently not a consequence of morphological differences or spatial segregation. Our data are consistent with the idea that female foraging strategies differed from male foraging strategies during incubation in ways that would be expected if females were still compensating for egg formation.
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The incredible mechanical strength and durability of mature fibrous tissues and their extremely limited turnover and regenerative capacity underscores the importance of proper matrix assembly during early postnatal growth. In tissues with composite extracellular matrix (ECM) structures, such as the adult knee meniscus, fibrous (Collagen-I rich), and cartilaginous (Collagen-II, proteoglycan-rich) matrix components are regionally segregated to the outer and inner portions of the tissue, respectively. While this spatial variation in composition is appreciated to be functionally important for resisting complex mechanical loads associated with gait, the establishment of these specialized zones is poorly understood. To address this issue, the following study tracked the growth of the murine meniscus from its embryonic formation through its first month of growth, encompassing the critical time-window during which animals begin to ambulate and weight bear. Using histological analysis, region specific high-throughput qPCR, and Col-1, and Col-2 fluorescent reporter mice, we found that matrix and cellular features defining specific tissue zones were already present at birth, before continuous weight-bearing had occurred. These differences in meniscus zones were further refined with postnatal growth and maturation, resulting in specialization of mature tissue regions. Taken together, this work establishes a detailed timeline of the concurrent spatiotemporal changes that occur at both the cellular and matrix level throughout meniscus maturation. The findings of this study provide a framework for investigating the reciprocal feedback between cells and their evolving microenvironments during assembly of a mechanically robust fibrocartilage tissue, thus providing insight into mechanisms of tissue degeneration and effective regenerative strategies.
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Cartilagem , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Menisco , Animais , Cartilagem/embriologia , Cartilagem/crescimento & desenvolvimento , Cartilagem/metabolismo , Diferenciação Celular , Proliferação de Células , Menisco/embriologia , Menisco/crescimento & desenvolvimento , Menisco/metabolismo , Camundongos , Camundongos TransgênicosRESUMO
Several recent studies have demonstrated that coculture of chondrocytes (CHs) with bone marrow-derived mesenchymal stem cells (MSCs) improves their chondrogenesis. This implies that intercellular communication dictates fate decisions in recipient cells and/or reprograms their metabolic state to support a differentiated function. While this coculture phenomenon is compelling, the differential chondroinductivity of zonal CHs on MSC cocultures, the nature of the molecular cargo, and their transport mechanisms remains undetermined. Here, we demonstrate that juvenile CHs in coculture with adult MSCs promote functional differentiation and improved matrix production. We further demonstrate that close proximity between the two cell types is a prerequisite for this response and that the outcome of this interaction improves viability, chondrogenesis, matrix formation, and homeostasis in the recipient MSCs. Furthermore, we visualized the transfer of intracellular contents from CHs to nearby MSCs and showed that inhibition of extracellular vesicle (EV) transfer blocks the synergistic effect of coculture, identifying EVs as the primary mode of communication in these cocultures. These findings will forward the development of therapeutic agents and more effective delivery systems to promote cartilage repair.
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Cartilagem/citologia , Cartilagem/fisiologia , Condrócitos/citologia , Condrócitos/fisiologia , Vesículas Extracelulares/fisiologia , Células-Tronco Mesenquimais/citologia , Animais , Bovinos , Comunicação Celular/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Condrogênese/fisiologia , Técnicas de Cocultura/métodos , Matriz Extracelular/fisiologia , Engenharia Tecidual/métodos , Alicerces TeciduaisRESUMO
Intervertebral disc (IVD) degeneration and associated back pain place a significant burden on the population. IVD degeneration is a progressive cascade of cellular, compositional, and structural changes, which results in a loss of disc height, disorganization of extracellular matrix architecture, tears in the annulus fibrosus which may involve herniation of the nucleus pulposus, and remodeling of the bony and cartilaginous endplates (CEP). These changes to the IVD often occur concomitantly, across the entire motion segment from the disc subcomponents to the CEP and vertebral bone, making it difficult to determine the causal initiating factor of degeneration. Furthermore, assessments of the subcomponents of the IVD have been largely qualitative, with most studies focusing on a single attribute, rather than multiple adjacent IVD substructures. The objective of this study was to perform a multiscale and multimodal analysis of human lumbar motion segments across various length scales and degrees of degeneration. We performed multiple assays on every sample and identified several correlations between structural and functional measurements of disc subcomponents. Our results demonstrate that with increasing Pfirrmann grade there is a reduction in disc height and nucleus pulposus T2 relaxation time, in addition to alterations in motion segment macromechanical function, disc matrix composition and cellular morphology. At the cartilage endplate-vertebral bone interface, substantial remodeling was observed coinciding with alterations in micromechanical properties. Finally, we report significant relationships between vertebral bone and nucleus pulposus metrics, as well as between micromechanical properties of the endplate and whole motion segment biomechanical parameters, indicating the importance of studying IVD degeneration as a whole organ.
Assuntos
Degeneração do Disco Intervertebral/fisiopatologia , Disco Intervertebral/fisiopatologia , Vértebras Lombares/fisiopatologia , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Disco Intervertebral/diagnóstico por imagem , Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/diagnóstico por imagem , Degeneração do Disco Intervertebral/patologia , Vértebras Lombares/diagnóstico por imagem , Vértebras Lombares/patologia , Masculino , Pessoa de Meia-Idade , Microtomografia por Raio-XRESUMO
Recent evidence has shown that, in addition to rigidity, the viscous response of the extracellular matrix (ECM) significantly affects the behavior and function of cells. However, the mechanism behind such mechanosensitivity toward viscoelasticity remains unclear. In this study, we systematically examined the dynamics of motor clutches (i.e., focal adhesions) formed between the cell and a viscoelastic substrate using analytical methods and direct Monte Carlo simulation. Interestingly, we observe that, for low ECM rigidity, maximum cell spreading is achieved at an optimal level of viscosity in which the substrate relaxation time falls between the timescale for clutch binding and its characteristic binding lifetime. That is, viscosity serves to stiffen soft substrates on a timescale faster than the clutch off-rate, which enhances cell-ECM adhesion and cell spreading. On the other hand, for substrates that are stiff, our model predicts that viscosity will not influence cell spreading, since the bound clutches are saturated by the elevated stiffness. The model was tested and validated using experimental measurements on three different material systems and explained the different observed effects of viscosity on each substrate. By capturing the mechanism by which substrate viscoelasticity affects cell spreading across a wide range of material parameters, our analytical model provides a useful tool for designing biomaterials that optimize cellular adhesion and mechanosensing.
Assuntos
Adesão Celular/fisiologia , Técnicas de Cultura de Células/instrumentação , Matriz Extracelular/química , Modelos Biológicos , Células 3T3 , Animais , Técnicas de Cultura de Células/métodos , Matriz Extracelular/metabolismo , Adesões Focais/metabolismo , Humanos , Hidrogéis , Integrinas/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Camundongos , Método de Monte Carlo , Reologia/métodos , Propriedades de Superfície , ViscosidadeRESUMO
Hydrogels are engineered with biochemical and biophysical signals to recreate aspects of the native microenvironment and to control cellular functions such as differentiation and matrix deposition. This deposited matrix accumulates within the pericellular space and likely affects the interactions between encapsulated cells and the engineered hydrogel; however, there has been little work to study the spatiotemporal evolution of matrix at this interface. To address this, metabolic labeling is employed to visualize the temporal and spatial positioning of nascent proteins and proteoglycans deposited by chondrocytes. Within covalently crosslinked hyaluronic acid hydrogels, chondrocytes deposit nascent proteins and proteoglycans in the pericellular space within 1 d after encapsulation. The accumulation of this matrix, as measured by an increase in matrix thickness during culture, depends on the initial hydrogel crosslink density with decreased thicknesses for more crosslinked hydrogels. Encapsulated fluorescent beads are used to monitor the hydrogel location and indicate that the emerging nascent matrix physically displaces the hydrogel from the cell membrane with extended culture. These findings suggest that secreted matrix increasingly masks the presentation of engineered hydrogel cues and may have implications for the design of hydrogels in tissue engineering and regenerative medicine.
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Hydrogels serve as valuable tools for studying cell-extracellular matrix interactions in three-dimensional environments that recapitulate aspects of native extracellular matrix. However, the impact of early protein deposition on cell behaviour within hydrogels has largely been overlooked. Using a bio-orthogonal labelling technique, we visualized nascent proteins within a day of culture across a range of hydrogels. In two engineered hydrogels of interest in three-dimensional mechanobiology studies-proteolytically degradable covalently crosslinked hyaluronic acid and dynamic viscoelastic hyaluronic acid hydrogels-mesenchymal stromal cell spreading, YAP/TAZ nuclear translocation and osteogenic differentiation were observed with culture. However, inhibition of cellular adhesion to nascent proteins or reduction in nascent protein remodelling reduced mesenchymal stromal cell spreading and nuclear translocation of YAP/TAZ, resulting in a shift towards adipogenic differentiation. Our findings emphasize the role of nascent proteins in the cellular perception of engineered materials and have implications for in vitro cell signalling studies and application to tissue repair.
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Linhagem da Célula , Hidrogéis/química , Células-Tronco Mesenquimais/citologia , Proteínas/metabolismo , Adesão Celular , Humanos , Mecanotransdução Celular , Transdução de SinaisRESUMO
Delivery of biofactors in a precise and controlled fashion remains a clinical challenge. Stimuli-responsive delivery systems can facilitate 'on-demand' release of therapeutics in response to a variety of physiologic triggering mechanisms (e.g. pH, temperature). However, few systems to date have taken advantage of mechanical inputs from the microenvironment to initiate drug release. Here, we developed mechanically-activated microcapsules (MAMCs) that are designed to deliver therapeutics in an on-demand fashion in response to the mechanically loaded environment of regenerating musculoskeletal tissues, with the ultimate goal of furthering tissue repair. To establish a suite of microcapsules with different thresholds for mechano-activation, we first manipulated MAMC physical dimensions and composition, and evaluated their mechano-response under both direct 2D compression and in 3D matrices mimicking the extracellular matrix properties and dynamic loading environment of regenerating tissue. To demonstrate the feasibility of this delivery system, we used an engineered cartilage model to test the efficacy of mechanically-instigated release of TGF-ß3 on the chondrogenesis of mesenchymal stem cells. These data establish a novel platform by which to tune the release of therapeutics and/or regenerative factors based on the physiologic dynamic mechanical loading environment, and will find widespread application in the repair and regeneration of numerous musculoskeletal tissues.
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The connective tissues of the musculoskeletal system can be grouped into fibrous, cartilaginous, and calcified tissues. While each tissue type has a distinct composition and function, the intersections between these tissues result in the formation of complex, composite, and graded junctions. The complexity of these interfaces is a critical aspect of their healthy function, but poses a significant challenge for their repair. In this review, we describe the organization and structure of complex musculoskeletal interfaces, identify emerging technologies for engineering such structures, and outline the requirements for assessing the complex nature of these tissues in the context of recapitulating their function through tissue engineering.
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Cartilagem Articular/fisiologia , Engenharia Tecidual/métodos , Alicerces Teciduais , Animais , Osso e Ossos/fisiologia , Humanos , Disco Intervertebral/patologia , Ligamentos/patologia , Articulação Temporomandibular/patologia , Tendões/patologiaRESUMO
The salient feature of anthropogenic climate change over the last century has been the rise in global mean temperature. However, global mean temperature is not used as an explanatory variable in studies of population-level response to climate change, perhaps because the signal-to-noise ratio of this gross measure makes its effect difficult to detect in any but the longest of datasets. Using a population of Leach's storm-petrels breeding in the Bay of Fundy, we tested whether local, regional, or global temperature measures are the best index of reproductive success in the face of climate change in species that travel widely between and within seasons. With a 56-year dataset, we found that annual global mean temperature (AGMT) was the single most important predictor of hatching success, more so than regional sea surface temperatures (breeding season or winter) and local air temperatures at the nesting colony. Storm-petrel reproductive success showed a quadratic response to rising temperatures, in that hatching success increased up to some critical temperature, and then declined when AGMT exceeded that temperature. The year at which AGMT began to consistently exceed that critical temperature was 1988. Importantly, in this population of known-age individuals, the impact of changing climate was greatest on inexperienced breeders: reproductive success of inexperienced birds increased more rapidly as temperatures rose and declined more rapidly after the tipping point than did reproductive success of experienced individuals. The generality of our finding that AGMT is the best predictor of reproductive success in this system may hinge on two things. First, an integrative global measure may be best for species in which individuals move across an enormous spatial range, especially within seasons. Second, the length of our dataset and our capacity to account for individual- and age-based variation in reproductive success increase our ability to detect a noisy signal.
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Aves/fisiologia , Mudança Climática , Reprodução/fisiologia , Temperatura , Animais , Estações do Ano , Fatores de TempoRESUMO
Treatment strategies to address pathologies of fibrocartilaginous tissue are in part limited by an incomplete understanding of structure-function relationships in these load-bearing tissues. There is therefore a pressing need to develop micro-engineered tissue platforms that can recreate the highly inhomogeneous tissue microstructures that are known to influence mechanotransductive processes in normal and diseased tissue. Here, we report the quantification of proteoglycan-rich microdomains in developing, ageing and diseased fibrocartilaginous tissues, and the impact of these microdomains on endogenous cell responses to physiologic deformation within a native-tissue context. We also developed a method to generate heterogeneous tissue-engineered constructs (hetTECs) with non-fibrous proteoglycan-rich microdomains engineered into the fibrous structure, and show that these hetTECs match the microstructural, micromechanical and mechanobiological benchmarks of native tissue. Our tissue-engineered platform should facilitate the study of the mechanobiology of developing, homeostatic, degenerating and regenerating fibrous tissues.
Assuntos
Sinalização do Cálcio , Condrócitos/metabolismo , Fibrocartilagem/metabolismo , Mecanotransdução Celular , Proteoglicanas/metabolismo , Estresse Mecânico , Adulto , Idoso , Animais , Bovinos , Células Cultivadas , Feminino , Fibrocartilagem/citologia , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Engenharia Tecidual , Suporte de CargaRESUMO
During mesenchymal development, the microenvironment gradually transitions from one that is rich in cell-cell interactions to one that is dominated by cell-ECM (extracellular matrix) interactions. Because these cues cannot readily be decoupled in vitro or in vivo, how they converge to regulate mesenchymal stem cell (MSC) mechanosensing is not fully understood. Here, we show that a hyaluronic acid hydrogel system enables, across a physiological range of ECM stiffness, the independent co-presentation of the HAVDI adhesive motif from the EC1 domain of N-cadherin and the RGD adhesive motif from fibronectin. Decoupled presentation of these cues revealed that HAVDI ligation (at constant RGD ligation) reduced the contractile state and thereby nuclear YAP/TAZ localization in MSCs, resulting in altered interpretation of ECM stiffness and subsequent changes in downstream cell proliferation and differentiation. Our findings reveal that, in an evolving developmental context, HAVDI/N-cadherin interactions can alter stem cell perception of the stiffening extracellular microenvironment.
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Caderinas/metabolismo , Adesão Celular , Fenômenos Mecânicos , Células-Tronco Mesenquimais/citologia , Animais , Fenômenos Biomecânicos , Bovinos , Adesão Celular/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Ácido Hialurônico/química , Ácido Hialurônico/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , MetilaçãoRESUMO
Given the significance of hydrogels as cell-instructive materials, it is important to understand how differences in their chemical and physical properties are able to direct cell fate. For example, it remains unclear how different hydrogel cross-linking chemistries and gelation mechanisms influence cell behavior. Here, we report on hyaluronan-tyramine (HA-Tyr) hydrogels prepared either with enzymatic cross-linking using horseradish peroxidase and H2O2 or with visible light (500 nm) triggered gelation. We demonstrate that when hydrogels are polymerized to equivalent Young's moduli, the specific cross-linking chemistry of HA-Tyr hydrogels can have a substantial impact on mesenchymal stem cell (MSC) behavior. MSCs cultured on HA-Tyr hydrogels exhibit increased cell spread areas on enzymatically formed substrates relative to photo-cross-linked matrices. While enzymatically formed hydrogels led to MSCs exhibiting greater cell focal adhesion length, MSCs cultured on the photo-cross-linked matrices exhibited smaller cell spread area and shorter focal adhesion length but generated increased traction stress. These findings highlight the importance of understanding hydrogel cross-linking chemistries when the role of biophysical cues in regulating stem cell fate is investigated.
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Ácido Hialurônico/química , Hidrogéis/química , Células-Tronco Mesenquimais/efeitos dos fármacos , Tiramina/química , Animais , Materiais Biocompatíveis/química , Bovinos , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Módulo de Elasticidade/efeitos dos fármacos , Peroxidase do Rábano Silvestre/metabolismo , Peróxido de Hidrogênio/metabolismo , Fenômenos MecânicosRESUMO
Biophysical stimuli presented to cells via microenvironmental properties (e.g., alignment and stiffness) or external forces have a significant impact on cell function and behavior. Recently, the cell nucleus has been identified as a mechanosensitive organelle that contributes to the perception and response to mechanical stimuli. However, the specific mechanotransduction mechanisms that mediate these effects have not been clearly established. Here, we offer a comprehensive review of the evidence supporting (and refuting) three hypothetical nuclear mechanotransduction mechanisms: physical reorganization of chromatin, signaling at the nuclear envelope, and altered cytoskeletal structure/tension due to nuclear remodeling. Our goal is to provide a reference detailing the progress that has been made and the areas that still require investigation regarding the role of nuclear mechanotransduction in cell biology. Additionally, we will briefly discuss the role that mathematical models of cell mechanics can play in testing these hypotheses and in elucidating how biophysical stimulation of the nucleus drives changes in cell behavior. While force-induced alterations in signaling pathways involving lamina-associated polypeptides (LAPs) (e.g., emerin and histone deacetylase 3 (HDAC3)) and transcription factors (TFs) located at the nuclear envelope currently appear to be the most clearly supported mechanism of nuclear mechanotransduction, additional work is required to examine this process in detail and to more fully test alternative mechanisms. The combination of sophisticated experimental techniques and advanced mathematical models is necessary to enhance our understanding of the role of the nucleus in the mechanotransduction processes driving numerous critical cell functions.