Adhesion Maturation Research Articles

Unveiling the Mechanotransduction Mechanism of Substrate Stiffness‐modulated Cancer Cell Motility via ROCK1 and ROCK2 Differentially Regulated Manner

In addition to soluble factors and stromal cells, the mechanical properties of extracellular matrix (ECM) are considered to be an important component of tumor microenvironment. In particular, substrate stiffness is a mechanical characteristic of the ECM by which certain anchorage cells sense and respond to a variety of cellular behaviors. At the same time, clinical studies have observed that substrate stiffness was increased during tumorigenesis, which could promote tumor malignant transformation and metastasis. However, the biomechanical behavior of tumor cells and the underlying mechanotransduction pathways remain unclear. Here, using stiffness‐regulable polyacrylamide (PAA) substrates to simulate the tissue stiffness at different progress stages of breast cancer in vitro, we found that moderate substrate stiffness induced the emergence of malignant phenotypes and further unregulated breast cancer cell motility. Subcellular structure observation showed that moderate substrate stiffness promote the formation of leading‐edge protrusion by accelerating focal adhesion maturation and polarizing intracellular traction force distribution. Moreover, the substrate stiffness directly activated integrin β1 and focal adhesion kinase (FAK), which accelerate focal adhesion (FA) maturation and induce the downstream cascades of intracellular signals of the RhoA/ROCK pathway. Interestingly, the differential regulatory mechanism between two ROCK isoforms (ROCK1 and ROCK2) in cell motility and mechanotransduction was clearly identified. ROCK1 phosphorylated the myosin‐regulatory light chain (MRLC) and facilitated the generation of traction force, while ROCK2 phosphorylated cofilin and regulated the cytoskeletal remodeling by suppressing F‐actin depolymerization. The ROCK isoforms differentially regulated the pathways of RhoA/ROCK1/p‐MLC and RhoA/ROCK2/p‐cofilin in a coordinate fashion to modulate breast cancer cell motility in a substrate stiffness‐dependent manner via integrin β1‐activated FAK signaling. Our findings provide new insights into the mechanisms of matrix mechanical property‐induced cancer cell migration and malignant behaviors. Taming these physical forces by implanting scaffolds with specific stiffness or by inhibiting specific ROCK isoforms could have potential implications for improving therapeutic outcomes in cancer.Support or Funding InformationThis work was supported, in part or in whole, by the National Natural Science Foundation of China (11772088, 31470906, 31700811, 11802056, and 31800780).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nanoneedle-Mediated Stimulation of Cell Mechanotransduction Machinery.

Biomaterial substrates can be engineered to present topographical signals to cells which, through interactions between the material and active components of the cell membrane, regulate key cellular processes and guide cell fate decisions. However, targeting mechanoresponsive elements that reside within the intracellular domain is a concept that has only recently emerged. Here, we show that mesoporous silicon nanoneedle arrays interact simultaneously with the cell membrane, cytoskeleton, and nucleus of primary human cells, generating distinct responses at each of these cellular compartments. Specifically, nanoneedles inhibit focal adhesion maturation at the membrane, reduce tension in the cytoskeleton, and lead to remodeling of the nuclear envelope at sites of impingement. The combined changes in actin cytoskeleton assembly, expression and segregation of the nuclear lamina, and localization of Yes-associated protein (YAP) correlate differently from what is canonically observed upon stimulation at the cell membrane, revealing that biophysical cues directed to the intracellular space can generate heretofore unobserved mechanosensory responses. These findings highlight the ability of nanoneedles to study and direct the phenotype of large cell populations simultaneously, through biophysical interactions with multiple mechanoresponsive components.

Heat-treated carbon coatings on poly (l-lactide) foils for tissue engineering

Carbon-based materials have emerged as promising candidates for a wide variety of biomedical applications, including tissue engineering. We have developed a simple but unique technique for patterning carbon-based substrates in order to control cell adhesion, growth and phenotypic maturation. Carbon films were deposited on PLLA foils from distances of 3 to 7 cm. Subsequent heat-treatment (60 °C, 1 h) created lamellar structures with dimensions decreasing from micro- to nanoscale with increasing deposition distance. All carbon films improved the spreading and proliferation of human osteoblast-like MG 63 cells, and promoted the alignment of these cells along the lamellar structures. Similar alignment was observed in human osteoblast-like Saos-2 cells and in human dermal fibroblasts. Type I collagen fibers produced by Saos-2 cells and fibroblasts were also oriented along the lamellar structures. These structures increased the activity of alkaline phosphatase in Saos-2 cells. Carbon coatings also supported adhesion and growth of vascular endothelial and smooth muscle cells, particularly flatter non-heated carbon films. On these films, the continuity of the endothelial cell layer was better than on heat-treated lamellar surfaces. Heat-treated carbon-coated PLLA is therefore more suitable for bone and skin tissue engineering, while carbon-coated PLLA without heating is more appropriate for vascular tissue engineering.

ROCK isoforms differentially modulate cancer cell motility by mechanosensing the substrate stiffness

Tumors are characterized by extracellular matrix (ECM) remodeling and stiffening. The importance of ECM stiffness in cancer is well known. However, the biomechanical behavior of tumor cells and the underlying mechanotransduction pathways remain unclear. Here, we used polyacrylamide (PAA) substrates to simulate tissue stiffness at different progress stages of breast cancer in vitro, and we observed that moderate substrate stiffness promoted breast cancer cell motility. The substrate stiffness directly activated integrin β1 and focal adhesion kinase (FAK), which accelerate focal adhesion (FA) maturation and induce the downstream cascades of intracellular signals of the RhoA/ROCK pathway. Interestingly, the differential regulatory mechanism between two ROCK isoforms (ROCK1 and ROCK2) in cell motility and mechanotransduction was clearly identified. ROCK1 phosphorylated the myosin regulatory light chain (MRLC) and facilitated the generation of traction force, while ROCK2 phosphorylated cofilin and regulated the cytoskeletal remodeling by suppressing F-actin depolymerization. The ROCK isoforms differentially regulated the pathways of RhoA/ROCK1/p-MLC and RhoA/ROCK2/p-cofilin in a coordinate fashion to modulate breast cancer cell motility in a substrate stiffness-dependent manner through integrin β1-activated FAK signaling. Our findings provide new insights into the mechanisms of matrix mechanical property-induced cancer cell migration and malignant behaviors. Statement of significanceHere, we examined the relationship between substrate stiffness and tumor cellular motility by using polyacrylamide (PAA) substrates to simulate the stages in vivo of breast cancer. The results elucidated the different regulatory roles between the two ROCK isoforms in cell motility and demonstrated that stiff substrate (38 kPa) mediated RhoA/ROCK1/p-MLC and RhoA/ROCK2/p-cofilin pathways through integrin β1-FAK activation and eventually promoted directional migration. Our discoveries would have significant implications in the understanding of the interaction between cancer cells and tumor microenvironments, and hence, it might provide new insights into the metastasis inhibition, which could be an adjuvant way of cancer therapy.

YAP and TAZ limit cytoskeletal and focal adhesion maturation to enable persistent cell motility.

Cell migration initiates by traction generation through reciprocal actomyosin tension and focal adhesion reinforcement, but continued motility requires adaptive cytoskeletal remodeling and adhesion release. Here, we asked whether de novo gene expression contributes to this cytoskeletal feedback. We found that global inhibition of transcription or translation does not impair initial cell polarization or migration initiation, but causes eventual migratory arrest through excessive cytoskeletal tension and over-maturation of focal adhesions, tethering cells to their matrix. The transcriptional coactivators YAP and TAZ mediate this feedback response, modulating cell mechanics by limiting cytoskeletal and focal adhesion maturation to enable persistent cell motility and 3D vasculogenesis. Motile arrest after YAP/TAZ ablation was partially rescued by depletion of the YAP/TAZ-dependent myosin phosphatase regulator, NUAK2, or by inhibition of Rho-ROCK-myosin II. Together, these data establish a transcriptional feedback axis necessary to maintain a responsive cytoskeletal equilibrium and persistent migration.

Mechanical Responses of Cancer Cells to Different Matrices Measured by AFM and FRET

Integrin-based focal adhesions matured at the front of the migrating cells and dissembled at the retracting edge of cells. In the formation, maturation and disassembly of focal adhesions, talin play an import role as a mediator between mechanosensitive membrane protein, integrin, and actin cytoskeleton. It is known that talin undergo conformational changes when they are stretched with higher force. Vinculin binding sites and actin binding domains is known to be exposed when talin are stretched. Matrix can impact cellular functions, such as cell growth and migration. However, how the physical properties of cancer cells change with the extracellular matrix remains to be an open question. Here, we measured the rigidities of cancer cells at different matrix using AFM and force transduction of cancer cells using a new Forster resonance energy transfer (FRET) tension sensor. Measurements the force changes on talin were conducted in normal cells (MCF10A), non-invasive cancerous cell (MCF7), and invasive cancerous human breast cells (MDA-MB-231). Using atomic force microscopy (AFM), rigidity-based mechanical properties of these three different cell lines under six different substrate stiffnesses are measured and quantified. Magnetic tweezers are used to apply force directly to integrins at focal adhesions. The correlation between the mechanical properties and forces on talin which are showed in our data reveal the different mechanisms adopted by normal and cancerous human breast cells. Our results indicate that the response of cancer cells to matrix is significantly different from that of normal cells, which provide an insight about the properties of cancer cells during the metastasis.

ARP3 Controls the Podocyte Architecture at the Kidney Filtration Barrier

SummaryPodocytes, highly specialized epithelial cells, build the outer part of the kidney filtration barrier and withstand high mechanical forces through a complex network of cellular protrusions. Here, we show that Arp2/3-dependent actin polymerization controls actomyosin contractility and focal adhesion maturation of podocyte protrusions and thereby regulates formation, maintenance, and capacity to adapt to mechanical requirements of the filtration barrier. We find that N-WASP-Arp2/3 define the development of complex arborized podocyte protrusions in vitro and in vivo. Loss of dendritic actin networks results in a pronounced activation of the actomyosin cytoskeleton and the generation of over-maturated but less efficient adhesion, leading to detachment of podocytes. Our data provide a model to explain podocyte protrusion morphology and their mechanical stability based on a tripartite relationship between actin polymerization, contractility, and adhesion.

P6 - Redox Modification of the Cytoskeleton During Cell Adhesion and Migration

Actin cytoskeleton assembly and organization are modulated by reactive oxygen species and the cellular redox environment. Focal adhesions link the cytoskeleton to the extracellular matrix and play an important role in growth and migration of vascular smooth muscle cells. We have previously shown that NADPH oxidase Nox4, a H2O2-producing enzyme, and its binding partner, polymerase delta interacting protein 2 (Poldip2), regulate turnover of focal adhesions. Downstream targets of Nox4 include Hic-5 and Hsp27, as well as the small GTPase, Rho A. Activation of Smad signaling by TGFp, which is known to strengthen focal adhesions, is Nox4-dependent, as is upregulation of Hsp27 and Hic-5 by TGFp. Similarly, overexpression of Poldip2 activates RhoA to prevent focal adhesion dissolution, while dominant-negative RhoA reverses the effects of Nox4/Poldip2 on focal adhesions. More recently, we discovered that H2O2 levels increase during integrin-mediated cell attachment due to activation of NOX4. Attachment induces F-actin oxidation by sulfenylation, and depletion of Poldip2 or NOX4 using siRNA, or scavenging of endogenous H2O2 with catalase, inhibits adhesion-induced F-actin oxidation. Of importance, the binding of F-actin to vinculin, a protein present in focal adhesion complexes that regulates focal adhesion maturation, is inhibited after transfection of an oxidation-resistant actin mutant. Silencing of Poldip2 or NOX4 also impairs actin-vinculin interaction. Taken together, our work has identified a major role for Poldip2/Nox4 in cytoskeletal organization, focal adhesion turnover and smooth muscle cell migration.

Fenofibrate Reduces the Asthma-Related Fibroblast-To-Myofibroblast Transition by TGF-Β/Smad2/3 Signaling Attenuation and Connexin 43-Dependent Phenotype Destabilization

The activation of human bronchial fibroblasts by transforming growth factor-β1 (TGF-β1) leads to the formation of highly contractile myofibroblasts in the process of the fibroblast–myofibroblast transition (FMT). This process is crucial for subepithelial fibrosis and bronchial wall remodeling in asthma. However, this process evades current therapeutic asthma treatment strategies. Since our previous studies showed the attenuation of the TGF-β1-induced FMT in response to lipid-lowering agents (e.g., statins), we were interested to see whether a corresponding effect could be obtained upon administration of hypolipidemic agents. In this study, we investigated the effect of fenofibrate on FMT efficiency in populations of bronchial fibroblasts derived from asthmatic patients. Fenofibrate exerted a dose-dependent inhibitory effect on the FMT, even though it did not efficiently affect the expression of α-smooth muscle actin (α-SMA; marker of myofibroblasts); however, it considerably reduced its incorporation into stress fibers through connexin 43 regulation. This effect was accompanied by disturbances in the actin cytoskeleton architecture, impairments in the maturation of focal adhesions, and the fenofibrate-induced deactivation of TGF-β1/Smad2/3 signaling. These data suggest that fenofibrate interferes with myofibroblastic differentiation during asthma-related subepithelial fibrosis. The data indicate the potential application of fenofibrate in the therapy and prevention of bronchial remodeling during the asthmatic process.

Cell cycle-dependent force transmission in cancer cells.

The generation of traction forces and their transmission to the extracellular environment supports the disseminative migration of cells from a primary tumor. In cancer cells, the periodic variation of nuclear stiffness during the cell cycle provides a functional link between efficient translocation and proliferation. However, the mechanical framework completing this picture remains unexplored. Here, the Fucci2 reporter was expressed in various human epithelial cancer cells to resolve their cell cycle phase transition. The corresponding tractions were captured by a recently developed reference-free confocal traction-force microscopy platform. The combined approach was conducive to the analysis of phase-dependent force variation at the level of individual integrin contacts. Detected forces were invariably higher in the G1 and early S phases than in the ensuing late S/G2, and locally colocalized with high levels of paxillin phosphorylation. Perturbation of paxillin phosphorylation at focal adhesions, obtained through the biochemical inhibition of focal adhesion kinase (FAK) or the transfection of nonphosphorylatable or phosphomimetic paxillin mutants, significantly diminished the force transmitted to the substrate. These data demonstrate a reproducible modulation of force transmission during the cell cycle progression of cancer cells, instrumental to their invasion of dense environments. In addition, they delineate a model in which paxillin phosphorylation supports the mechanical maturation of adhesions relaying forces to the substrate.

Abstract A66: The tissue transglutaminase 2–fibronectin protein complex: A new target in ovarian cancer

Abstract Here we describe a small-molecule inhibitor (SMI) that disrupts the tissue transglutaminase 2 (TG2)-fibronectin (FN) interaction. TG2 is a multifunctional protein, with enzymatic (transglutaminase, isopeptidase, protein disulfide isomerase), GTPase, and extracellular matrix (ECM) scaffold activities. TG2 directly interacts with FN through its N-terminus domain, playing an important role in stabilizing ternary TG2/FN/integrin complexes, which regulate cell adhesion to the matrix. Our group has previously shown that TG2 overexpressed in ovarian cancer (OC) promotes epithelial-to-mesenchymal transition (EMT) contributing to OC cell invasiveness and metastatic behavior. We showed that the interaction with FN is critical to these functions. Proximity ligation assay was used to measure TG2/integrin beta 1 interaction in OC tumors and identified TG2/integrin beta 1 complex in >50% of ovarian tumors, irrespective of histologic type. Based on those findings, we conducted the high-throughput screening and identified TG53 as the lead TG2/FN inhibitory compound. Structure-activity relationship-based approach was used to optimize the physicochemical properties of TG53, leading to design and chemical synthesis of 6 analogues (MT1-6). All analogues blocked TG2/FN interaction, as measured by an ELISA assay, and OC cell adhesion to FN, as measured by a solid phase assay, MT4 being the most potent inhibitor. MT4 downregulates integrin-initiated signaling in OC cells, leading to downstream inhibition of FAK and ERK. Immunofluorescence staining demonstrated that the actin cytoskeleton adopts a cortical localization pattern in MT4-treated cells. MT4 caused membrane ruffling delaying the formation of stable focal contacts and mature adhesions points. Pretreatment with MT4 sensitized OC cells to paclitaxel, as measured by colony-forming assay. In an intraperitoneal OC mouse model, MT4 caused decreased OC cell adhesion to the peritoneum, inhibiting peritoneal dissemination. Hence, MT4 is a potential candidate to be developed and used in combination with current chemotherapeutics to target OC tumor progression. Further studies will continue to optimize this new class of TG2/FN SMIs for potential clinical use in OC. Citation Format: Livia Elena Sima, Bakhtiyor Yakubov, Salvatore Condello, Horacio Cardenas, Zhong Yin Zhang, Lan Chen, Daniela Elena Matei. The tissue transglutaminase 2–fibronectin protein complex: A new target in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A66.

A micron-scale surface topography design reducing cell adhesion to implanted materials

The micron-scale surface topography of implanted materials represents a complementary pathway, independent of the material biochemical properties, regulating the process of biological recognition by cells which mediate the inflammatory response to foreign bodies. Here we explore a rational design of surface modifications in micron range to optimize a topography comprised of a symmetrical array of hexagonal pits interfering with focal adhesion establishment and maturation. When implemented on silicones and hydrogels in vitro, the anti-adhesive topography significantly reduces the adhesion of macrophages and fibroblasts and their activation toward effectors of fibrosis. In addition, long-term interaction of the cells with anti-adhesive topographies markedly hampers cell proliferation, correlating the physical inhibition of adhesion and complete spreading with the natural progress of the cell cycle. This solution for reduction in cell adhesion can be directly integrated on the outer surface of silicone implants, as well as an additive protective conformal microstructured biocellulose layer for materials that cannot be directly microstructured. Moreover, the original geometry imposed during manufacturing of the microstructured biocellulose membranes are fully retained upon in vivo exposure, suggesting a long lasting performance of these topographical features after implantation.

Paxillin phosphorylation at serine 273 and its effects on Rac, Rho and adhesion dynamics.

Focal adhesions are protein complexes that anchor cells to the extracellular matrix. During migration, the growth and disassembly of these structures are spatiotemporally regulated, with new adhesions forming at the leading edge of the cell and mature adhesions disassembling at the rear. Signalling proteins and structural cytoskeletal components tightly regulate adhesion dynamics. Paxillin, an adaptor protein within adhesions, is one of these proteins. Its phosphorylation at serine 273 (S273) is crucial for maintaining fast adhesion assembly and disassembly. Paxillin is known to bind to a GIT1-βPIX-PAK1 complex, which increases the local activation of the small GTPase Rac. To understand quantitatively the behaviour of this system and how it relates to adhesion assembly/disassembly, we developed a mathematical model describing the dynamics of the small GTPases Rac and Rho as determined by paxillin S273 phosphorylation. Our model revealed that the system possesses bistability, where switching between uninduced (active Rho) and induced (active Rac) states can occur through a change in rate of paxillin phosphorylation or PAK1 activation. The bistable switch is characterized by the presence of memory, minimal change in the levels of active Rac and Rho within the induced and uninduced states, respectively, and the limited regime of monostability associated with the uninduced state. These results were validated experimentally by showing the presence of bimodality in adhesion assembly and disassembly rates, and demonstrating that Rac activity increases after treating Chinese Hamster Ovary cells with okadaic acid (a paxillin phosphatase inhibitor), followed by a modest recovery after 20 min washout. Spatial gradients of phosphorylated paxillin in a reaction-diffusion model gave rise to distinct regions of Rac and Rho activities, resembling polarization of a cell into front and rear. Perturbing several parameters of the model also revealed important insights into how signalling components upstream and downstream of paxillin phosphorylation affect dynamics.

In vitro response of mesenchymal stem cells to biomimetic hydroxyapatite substrates: A new strategy to assess the effect of ion exchange

Biomaterials can interact with cells directly, that is, by direct contact of the cells with the material surface, or indirectly, through soluble species that can be released to or uptaken from the surrounding fluids. However, it is difficult to characterise the relevance of this fluid-mediated interaction separately from the topography and composition of the substrate, because they are coupled variables. These fluid-mediated interactions are amplified in the case of highly reactive calcium phosphates (CaPs) such as biomimetic calcium deficient hydroxyapatite (CDHA), particularly in static in vitro cultures. The present work proposes a strategy to decouple the effect of ion exchange from topographical features by adjusting the volume ratio between the cell culture medium and biomaterial (VCM/VB). Increasing this ratio allowed mitigating the drastic ionic exchanges associated to the compositional changes experienced by the material exposed to the cell culture medium. This strategy was validated using rat mesenchymal stem cells (rMSCs) cultured on CDHA and beta-tricalcium phosphate (β-TCP) discs using different VCM/VB ratios. Whereas in the case of β-TCP the cell response was not affected by this ratio, a significant effect on cell adhesion and proliferation was found for the more reactive CDHA. The ionic exchange, produced by CDHA at low VCM/VB, altered cell adhesion due to the reduced number of focal adhesions, caused cell shrinkage and further rMCSs apoptosis. This was mitigated when using a high VCM/VB, which attenuated the changes of calcium and phosphate concentrations in the cell culture medium, resulting in rMSCs spreading and a viability over time. Moreover, rMSCs showed an earlier expression of osteogenic genes on CDHA compared to sintered β-TCP when extracellular calcium fluctuations were reduced. Statement of SignificanceFluid mediated interactions play a significant role in the bioactivity of calcium phosphates. Ionic exchange is amplified in the case of biomimetic hydroxyapatite, which makes the in vitro characterisation of cell-material interactions especially challenging. The present work proposes a novel and simple strategy to explore the mechanisms of interaction of biomimetic and sintered calcium phosphates with mesenchymal stem cells. The effects of topography and ion exchange are analysed separately by modifying the volume ratio between cell culture medium and biomaterial. High ionic fluctuations interfered in the maturation of focal adhesions, hampering cell adhesion and leading to increased apoptosis and reduced proliferation rate.

Substrate micropatterns produced by polymer demixing regulate focal adhesions, actin anisotropy, and lineage differentiation of stem cells

Stem cells are adherent cells whose multipotency and differentiation can be regulated by numerous microenvironmental signals including soluble growth factors and surface topography. This study describes a simple method for creating distinct micropatterns via microphase separation resulting from polymer demixing of poly(desaminotyrosyl-tyrosine carbonate) (PDTEC) and polystyrene (PS). Substrates with co-continuous (ribbons) or discontinuous (islands and pits) PDTEC regions were obtained by varying the ratio of PDTEC and sacrificial PS. Human mesenchymal stem cells (MSCs) cultured on co-continuous PDTEC substrates for 3 days in bipotential adipogenic/osteogenic (AD/OS) induction medium showed no change in cell morphology but exhibited increased anisotropic cytoskeletal organization and larger focal adhesions when compared to MSCs cultured on discontinuous micropatterns. After 14 days in bipotential AD/OS induction medium, MSCs cultured on co-continuous micropatterns exhibited increased expression of osteogenic markers, whereas MSCs on discontinuous PDTEC substrates showed a low expression of adipogenic and osteogenic differentiation markers. Substrates with graded micropatterns were able to reproduce the influence of local underlying topography on MSC differentiation, thus demonstrating their potential for high throughput analysis. This work presents polymer demixing as a simple, non-lithographic technique to produce a wide range of micropatterns on surfaces with complex geometries to influence cellular and tissue regenerative responses. Statement of SignificanceA better understanding of how engineered microenvironments influence stem cell differentiation is integral to increasing the use of stem cells and materials in a wide range of tissue engineering applications. In this study, we show the range of topography obtained by polymer demixing is sufficient for investigating how surface topography affects stem cell morphology and differentiation. Our findings show that co-continuous topographies favor early (3-day) cytoskeletal anisotropy and focal adhesion maturation as well as long-term (14-day) expression of osteogenic differentiation markers. Taken together, this study presents a simple approach to pattern topographies that induce divergent responses in stem cell morphology and differentiation.

Mechanically robust cryogels with injectability and bioprinting supportability for adipose tissue engineering

Bioengineered adipose tissues have gained increased interest as a promising alternative to autologous tissue flaps and synthetic adipose fillers for soft tissue augmentation and defect reconstruction in clinic. Although many scaffolding materials and biofabrication methods have been investigated for adipose tissue engineering in the last decades, there are still challenges to recapitulate the appropriate adipose tissue microenvironment, maintain volume stability, and induce vascularization to achieve long-term function and integration. In the present research, we fabricated cryogels consisting of methacrylated gelatin, methacrylated hyaluronic acid, and 4arm poly(ethylene glycol) acrylate (PEG-4A) by using cryopolymerization. The cryogels were repeatedly injectable and stretchable, and the addition of PEG-4A improved the robustness and mechanical properties. The cryogels supported human adipose progenitor cell (HWA) and adipose derived mesenchymal stromal cell adhesion, proliferation, and adipogenic differentiation and maturation, regardless of the addition of PEG-4A. The HWA laden cryogels facilitated the co-culture of human umbilical vein endothelial cells (HUVEC) and capillary-like network formation, which in return also promoted adipogenesis. We further combined cryogels with 3D bioprinting to generate handleable adipose constructs with clinically relevant size. 3D bioprinting enabled the deposition of multiple bioinks onto the cryogels. The bioprinted flap-like constructs had an integrated structure without delamination and supported vascularization. Statement of SignificanceAdipose tissue engineering is promising for reconstruction of soft tissue defects, and also challenging for restoring and maintaining soft tissue volume and shape, and achieving vascularization and integration. In this study, we fabricated cryogels with mechanical robustness, injectability, and stretchability by using cryopolymerization. The cryogels promoted cell adhesion, proliferation, and adipogenic differentiation and maturation of human adipose progenitor cells and adipose derived mesenchymal stromal cells. Moreover, the cryogels also supported 3D bioprinting on top, forming vascularized adipose constructs. This study demonstrates the potential of the implementation of cryogels for generating volume-stable adipose tissue constructs and provides a strategy to fabricate vascularized flap-like constructs for complex soft tissue regeneration.

Carbon nanotube multilayered nanocomposites as multifunctional substrates for actuating neuronal differentiation and functions of neural stem cells

Carbon nanotubes (CNTs) have shown potential applications in neuroscience as growth substrates owing to their numerous unique properties. However, a key concern in the fabrication of homogeneous composites is the serious aggregation of CNTs during incorporation into the biomaterial matrix. Moreover, the regulation mechanism of CNT-based substrates on neural differentiation remains unclear. Here, a novel strategy was introduced for the construction of CNT nanocomposites via layer-by-layer assembly of negatively charged multi-walled CNTs and positively charged poly(dimethyldiallylammonium chloride). Results demonstrated that the CNT-multilayered nanocomposites provided a potent regulatory signal over neural stem cells (NSCs), including cell adhesion, viability, differentiation, neurite outgrowth, and electrophysiological maturation of NSC-derived neurons. Importantly, the dynamic molecular mechanisms in the NSC differentiation involved the integrin-mediated interactions between NSCs and CNT multilayers, thereby activating focal adhesion kinase, subsequently triggering downstream signaling events to regulate neuronal differentiation and synapse formation. This study provided insights for future applications of CNT-multilayered nanomaterials in neural fields as potent modulators of stem cell behavior.

Acidic pHe regulates cytoskeletal dynamics through conformational integrin β1 activation and promotes membrane protrusion

An acidic extracellular pH (pHe) in the tumor microenvironment has been suggested to facilitate tumor growth and metastasis. However, the molecular mechanisms by which tumor cells sense acidic signal to induce a transition to an aggressive phenotype remain elusive. Here, we showed that an acidic pHe (pH 6.5) stimulation resulted in protrusion and epithelial-mesenchymal transition (EMT) of cancer cells, which promoted migration and matrix degeneration. Using computational molecular dynamics simulations, we reported acidic pHe-induced opening of the Integrin dimers (α5β1) headpiece which indicated the activation of integrin. Moreover, acidic pHe promoted maturation of focal adhesions, temporal activation of Rho GTPases and microfilament reorganization through integrin β1-activated FAK signaling. Furthermore, mechanical balance of cytoskeleton (actin, tubulin and vimentin) contributed to acidic pHe-triggered protrusion and morphology change. Taken together, these findings revealed that integrin β1 could be a novel pH-regulated sensitive molecule which confers protrusion and malignant phenotype of cancer cells.

An automated quantitative analysis of cell, nucleus and focal adhesion morphology.

Adherent cells sense the physical properties of their environment via focal adhesions. Improved understanding of how cells sense and response to their physical surroundings is aided by quantitative evaluation of focal adhesion size, number, orientation, and distribution in conjunction with the morphology of single cells and the corresponding nuclei. We developed a fast, user-friendly and automated image analysis algorithm capable of capturing and characterizing these individual components with a high level of accuracy. We demonstrate the robustness and applicability of the algorithm by quantifying morphological changes in response to a variety of environmental changes as well as manipulations of cellular components of mechanotransductions. Finally, as a proof-of-concept we use our algorithm to quantify the effect of Rho-associated kinase inhibitor Y-27632 on focal adhesion maturation. We show that a decrease in cell contractility leads to a decrease in focal adhesion size and aspect ratio.

Surface nanoporosity has a greater influence on osteogenic and bacterial cell adhesion than crystallinity and wettability

There has been much emphasis on the influence of crystallinity and wettability for modulating cell activity, particularly for bone biomaterials. In this context, we have generated titanium oxide layers with similar mesoporous topography and surface roughness but with amorphous or crystalline oxide layers and differential wettability. We then investigated their influence on the behavior of MC3T3 osteoblastic and bacterial cells. There was no difference in cell adhesion, spreading and growth on amorphous and crystalline surfaces. The number of focal adhesions was similar, however, cells on the amorphous surface exhibited a higher frequency of mature adhesions. The crystallinity of the surface layers also had no bearing on bacterial adhesion. While it cannot be excluded that surface crystallinity, roughness and wettability contribute to some degree to determining cell behavior, our data suggest that physical characteristics of surfaces represent the major determinant.