Development of a prognostic model for lung adenocarcinoma polarity-related genes and analysis of immune landscape.

Despite the progress made in the management of lung adenocarcinoma (LUAD), the overall prognosis for LUAD individuals remains suboptimal. While the role of cell polarity in tumor invasion and metastasis is well established, its prognostic significance in LUAD is still unknown. Differential analysis was performed on the Cancer Genome Atlas (TCGA)-LUAD and normal lung tissue, and candidate genes were identified by intersecting differentially expressed genes with polarity-related genes (PRGs). A prognostic model was constructed using univariate and multivariate Cox regression and LASSO regression. To enhance the robustness of the analysis, an independent prognostic analysis was conducted by incorporating relevant clinical information. The accuracy and sensitivity of the model were validated using survival analysis and ROC curves. Finally, immune landscape, immune therapy, tumor mutation burden, and drug sensitivity analysis were carried out on high- and low-risk patients. Ten prognostic genes were screened to divide LUAD patients into different risk groups. Survival analysis, ROC curves, and univariate/multivariate Cox regression analyses collectively demonstrated the favorable predictive performance of the model, which could be an independent prognostic factor. The nomogram, in conjunction with the calibration curve, demonstrated the model's compelling predictive capacity in prognosticating the overall survival of LUAD individuals. Low-risk LUAD patients exhibited heightened levels of immune cell infiltration, immune scores, and immune checkpoint expression compared to high-risk individuals. So, they may have a greater likelihood of benefiting from immune therapy. The high-risk group demonstrated a remarkably higher tumor mutation burden (TMB) in contrast with the low-risk group. XAV-939, Fulvestrant, and SR16157 may have potential value in the clinical use of LUAD. We revealed the potential linkage between PRGs and LUAD prognosis, and the application of these prognostic factors in risk stratification and prognosis prediction of LUAD patients may be of great significance.

Mesenchymal stem cells-derived extracellular vesicles containing miR-378a-3p inhibit the occurrence of inflammatory bowel disease by targeting GATA2.

This study sought to determine whether mesenchymal stem cells-derived extracellular vesicles (MSCs-EVs) carrying microRNA-378a-3p (miR-378a-3p) could affect the pathogenesis of inflammatory bowel disease (IBD) by regulating the GATA-binding protein 2 (GATA2)/aquaporin-4 (AQP4)/peroxisome proliferator-activated receptor α (PPAR-α) axis. Initially, colon mucosa biopsy tissues were harvested from healthy controls and patients with IBD for qRT-PCR and immunohistochemistry analysis. EVs harvested from MSCs and lipopolysaccharide (LPS) were used to stimulate the M064 cells to establish an in vitro inflammation cell model. Besides, 2,4,6-trinitrobenzene sulfonic acid intracolon administration was performed to establish in vivo IBD mouse models. After loss- and gain-of-function assays, the regulatory role of MSCs-derived EVs loaded with manipulated miR-378a-3p in IBD in relation to GATA2/AQP4/PPAR-α were explored. Upregulation of GATA2 was identified in the colon tissue of IBD patients. GATA2, which was a target gene of miR-378a-3p, transcriptionally upregulated AQP4. After silencing of GATA2, LPS-induced apoptosis of M064 cells was reduced by the downregulation of AQP4. Decreased AQP4 contributed to PPAR-α pathway inactivation and weakened the LPS-induced apoptosis of M064 cells. MSCs-EVs delivering miR-378a-3p suppressed the GATA2/AQP4/PPAR-α pathway, which reduced LPS-induced apoptosis of M064 cells and the occurrence of IBD in mice. Altogether, the current study illustrated that MSCs-EVs transfer miR-378a-3p to reduce the GATA2 expression, which downregulates AQP4 to block the PPAR-α signalling pathway, thus suppressing the occurrence of IBD.

Recombinant Human Proteoglycan 4 (rhPRG4) Downregulates TNFα-Stimulated NFκB Activity and FAT10 Expression in Human Corneal Epithelial Cells.

Dry Eye Disease (DED) is a complex pathology affecting millions of people with significant impact on quality of life. Corneal inflammation, including via the nuclear factor kappa B (NFκB) pathway, plays a key etiological role in DED. Recombinant human proteoglycan 4 (rhPRG4) has been shown to be a clinically effective treatment for DED that has anti-inflammatory effects in corneal epithelial cells, but the underlying mechanism is still not understood. Our goal was to understand if rhPRG4 affects tumor necrosis factor α (TNFα)-stimulated inflammatory activity in corneal epithelial cells. We treated hTERT-immortalized corneal epithelial (hTCEpi) cells ± TNFα ± rhPRG4 and performed Western blotting on cell lysate and RNA sequencing. Bioinformatics analysis revealed that rhPRG4 had a significant effect on TNFα-mediated inflammation with potential effects on matricellular homeostasis. rhPRG4 reduced activation of key inflammatory pathways and decreased expression of transcripts for key inflammatory cytokines, interferons, interleukins, and transcription factors. TNFα treatment significantly increased phosphorylation and nuclear translocation of p65, and rhPRG4 significantly reduced both these effects. RNA sequencing identified human leukocyte antigen (HLA)-F adjacent transcript 10 (FAT10), a ubiquitin-like modifier protein which has not been studied in the context of DED, as a key pro-inflammatory transcript increased by TNFα and decreased by rhPRG4. These results were confirmed at the protein level. In summary, rhPRG4 is able to downregulate NFκB activity in hTCEpi cells, suggesting a potential biological mechanism by which it may act as a therapeutic for DED.

Peroxo Species Formed in the Bulk of Silicate Cathodes.

Oxygen redox in Li-rich oxides may boost the energy density of lithium-ion batteries by incorporating oxygen chemistry in solid cathodes. However, oxygen redox in the bulk usually entangles with voltage hysteresis and oxygen release, resulting in a prolonged controversy in literature on oxygen transformation. Here, we report spectroscopic evidence of peroxo species formed and confined in silicate cathodes amid oxygen redox at high voltage, accompanied by Co2+ /Co3+ redox dominant at low voltage. First-principles calculations reveal that localized electrons on dangling oxygen drive the O-O dimerization. The covalence between the binding cation and the O-O dimer determines the degree of electron transfer in oxygen transformation. Dimerization induces irreversible structural distortion and slow kinetics. But peroxo formation can minimize the voltage drop and volume expansion in cumulative cationic and anionic redox. These findings offer insights into oxygen redox in the bulk for the rational design of high-energy-density cathodes.

Nanostructure and its effect on electrochemical properties of polyanionic Li2CoSiO4 for lithium ion batteries.

This work reports a facile strategy to synthesize carbon-coated Li2CoSiO4/C particles with rich nanostructures by a two-step scheme starting with a low-temperature hydrothermal method. The size and morphology of particle aggregates can be regulated by the (OH-) concentration and viscosity of the precursor solution, a mixture of ethylene glycol and deionized water, for the hydrothermal synthesis. In addition to the good electrical conductivity from the carbon coating, the size of the primary nanoparticles and the mesopore associated with the aggregations play an important role in improving the electrochemical properties of polyanionic Li2CoSiO4. The low-dimensional belt-like and sheet-like Li2CoSiO4/C nanomaterials present a higher reversible capacity 136.6 mAh g-1 and 147 mAh g-1, respectively, in the first charging-discharging cycle between 2 V and 4.6 V. XRD, SEM/TEM, and EDS are used to characterize the crystalline structure and aggregation patterns. TGA and Raman spectra are employed to analyze the carbon coating on different morphologies. The analysis of electrochemical impedance spectroscopy highlights the critical role of the interface between the electrolyte and particles. This study provides insights into the rational design and synthesis of high-performance polyanionic cathodes including silicates.

Dimension-Controllable Microtube Arrays by Dynamic Holographic Processing as 3D Yeast Culture Scaffolds for Asymmetrical Growth Regulation.

Transparent microtubes can function as unique cell culture scaffolds, because the tubular 3D microenvironment they provide is very similar to the narrow space of capillaries in vivo. However, how to realize the fabrication of microtube-arrays with variable cross-section dynamically remains challenging. Here, a dynamic holographic processing method for producing high aspect ratio (≈20) microtubes with tunable outside diameter (6-16 µm) and inside diameter (1-10 µm) as yeast culture scaffolds is reported. A ring-structure Bessel beam is modulated from a typical Gaussian-distributed femtosecond laser beam by a spatial light modulator. By combining the axial scanning of the focused beam and the dynamic display of holograms, dimension-controllable microtube arrays (straight, conical, and drum-shape) are rapidly produced by two-photon polymerization. The outside and inside diameters, tube heights, and spatial arrangements are readily tuned by loading different computer-generated holograms and changing the processing parameters. The transparent microtube array as a nontrivial tool for capturing and culturing the budding yeasts reveals the significant effect of tube diameter on budding characteristics. In particular, the conical tube with the inside diameter varying from 5 to 10 µm has remarkable asymmetrical regulation on the growth trend of captured yeasts.

Arch-like microsorters with multi-modal and clogging-improved filtering functions by using femtosecond laser multifocal parallel microfabrication.

Conventional micropore membranes based size sorting have been widely applied for single-cell analysis. However, only a single filtering size can be achieved and the clogging issue cannot be completely avoided. Here, we propose a novel arch-like microsorter capable of multimodal (high-, band- and low-capture mode) sorting of particles. The target particles can pass through the front filter and are then trapped by the back filter, while the non-target particles can bypass or pass through the microsorter. This 3D arch-like microstructures are fabricated inside a microchannel by femtosecond laser parallel multifocal scanning. The designed architecture allows for particles isolation free of clogging over 20 minutes. Finally, as a proof of concept demonstration, SUM159 breast cancer cells are successfully separated from whole blood.

High efficiency fabrication of complex microtube arrays by scanning focused femtosecond laser Bessel beam for trapping/releasing biological cells.

In this paper, we present a focused femtosecond laser Bessel beam scanning technique for the rapid fabrication of large-area 3D complex microtube arrays. The femtosecond laser beam is converted into several Bessel beams by two-dimensional phase modulation using a spatial light modulator. By scanning the focused Bessel beam along a designed route, microtubes with variable size and flexible geometry are rapidly fabricated by two-photon polymerization. The fabrication time is reduced by two orders of magnitude in comparison with conventional point-to-point scanning. Moreover, we construct an effective microoperating system for single cell manipulation using microtube arrays, and demonstrate its use in the capture, transfer, and release of embryonic fibroblast mouse cells as well as human breast cancer cells. The new fabrication strategy provides a novel method for the rapid fabrication of functional devices using a flexibly tailored laser beam.

Stable and Oscillatory Hypoxia Differentially Regulate Invasibility of Breast Cancer Associated Fibroblasts.

As local regions in the tumor outstrip their oxygen supply, hypoxia can develop, affecting not only the cancer cells, but also other cells in the microenvironment, including cancer associated fibroblasts (CAFs). Hypoxia is also not necessarily stable over time, and can fluctuate or oscillate. Hypoxia Inducible Factor-1 is the master regulator of cellular response to hypoxia, and can also exhibit oscillations in its activity. To understand how stable, and fluctuating hypoxia influence breast CAFs, we measured changes in gene expression in CAFs in normoxia, hypoxia, and oscillatory hypoxia, as well as measured change in their capacity to resist, or assist breast cancer invasion. We show that hypoxia has a profound effect on breast CAFs causing activation of key pathways associated with fibroblast activation, but reduce myofibroblast activation and traction force generation. We also found that oscillatory hypoxia, while expectedly resulted in a "sub-hypoxic" response in gene expression, it resulted in specific activation of pathways associated with actin polymerization and actomyosin maturation. Using traction force microscopy, and a nanopatterned stromal invasion assay, we show that oscillatory hypoxia increases contractile force generation vs stable hypoxia, and increases heterogeneity in force generation response, while also additively enhancing invasibility of CAFs to MDA-MB-231 invasion. Our data show that stable and unstable hypoxia can regulate many mechnobiological characteristics of CAFs, and can contribute to transformation of CAFs to assist cancer dissemination and onset of metastasis.

Oscillatory Hypoxia Induced Unfolded Protein Folding Response Gene Expression Predicts Low Survival in Human Breast Cancer Patients.

Hypoxia is one of the key factors in the tumor microenvironment regulating nearly all steps in the metastatic cascade in many cancers, including in breast cancer. The hypoxic regions can however be dynamic with the availability of oxygen fluctuating or oscillating. The canonical response to hypoxia is relayed by transcription factor HIF-1, which is stabilized in hypoxia and acts as the master regulator of a large number of downstream genes. However, HIF-1 transcriptional activity can also fluctuate either due to unstable hypoxia, or by lactate mediated non-canonical degradation of HIF-1. Our understanding of how oscillatory hypoxia or HIF-1 activity specifically influence cancer malignancy is very limited. Here, using MDA-MB-231 cells as a model of triple negative breast cancer characterized by severe hypoxia, we measured the gene expression changes induced specifically by oscillatory hypoxia. We found that oscillatory hypoxia can specifically regulate gene expression differently, and at times opposite to stable hypoxia. Using The Cancer Genome Atlas (TCGA) RNAseq data of human cancer samples, we show that the oscillatory specific gene expression signature in MDA-MB-231 is enriched in most human cancers, and prognosticate low survival in breast cancer patients. In particular, we found that oscillatory hypoxia, unlike stable hypoxia, induces unfolded protein folding response (UPR) in cells resulting in gene expression predicting reduced survival.

Oscillatory Hypoxia Can Induce Senescence of Adipose-Derived Mesenchymal Stromal Cells Potentiating Invasive Transformation of Breast Epithelial Cells.

Obesity is strongly associated with occurrence, metastasis, and resistance to therapy in breast cancers, which also exhibit high adipose content in the tumor microenvironment. Adipose tissue-derived mesenchymal stromal cells (ASCs) are recruited to breast cancer by many mechanisms, including hypoxia, and contribute to metastatic transition of the cancer. Breast cancers are characterized by regions of hypoxia, which can be temporally unstable owing to a mismatch between oxygen supply and consumption. Using a high-sensitivity nanopatterned stromal invasion assay, we found that ASCs could promote stromal invasion of not only breast cancer cell lines but also MCF10A1, a cell line derived from untransformed breast epithelium. RNA sequencing of MCF10A1 cells conditioned with medium from ASCs revealed upregulation of genes associated with increased cell migration, chemotaxis, and metastasis. Furthermore, we found that fluctuating or oscillating hypoxia could induce senescence in ASCs, which could result in an increased invasive potential in the treated MCF10A1 cells. These findings highlight the complex interplay within the breast cancer microenvironment, hypoxia, and the role of ASCs in transforming even non-cancerous breast epithelium toward an invasive phenotype, providing insights into early metastatic events.

Contact Guidance Drives Upward Cellular Migration at the Mesoscopic Scale.

Introduction: Cancer metastasis is associated with increased cancer incidence, recurrence, and mortality. The role of cell contact guidance behaviors in cancer metastasis has been recognized but has not been elucidated yet. Methods: The contact guidance behavior of cancer cells in response to topographical constraints is identified using microgrooved substrates with varying dimensions at the mesoscopic scale. Then, the cell morphology is determined to quantitatively analyze the effects of substrate dimensions on cells contact guidance. Cell density and migrate velocity signatures within the cellular population are determined using time-lapse phase-contrast microscopy. The effect of soluble factors concentration is determined by culturing cells upside down. Then, the effect of cell-substrate interaction on cell migration is investigated using traction force microscopy. Results: With increasing depth and decreasing groove width, cell elongation and alignment are enhanced, while cell spreading is inhibited. Moreover, cells display preferential distribution on the ridges, which is found to be more pronounced with increasing depth and groove width. Determinations of cell density and migration velocity signatures reveal that the preferential distribution on ridges is caused by cell upward migration. Combined with traction force measurement, we find that migration toward ridges is governed by different cell-substrate interactions between grooves and ridges caused by geometrical constraints. Interestingly, the upward migration of cells at the mesoscopic scale is driven by entropic maximization. Conclusions: The mesoscopic cell contact guidance mechanism based on the entropic force driven theory provides basic support for the study of cell alignment and migration along healthy tissues with varying size, thereby aiding in the prediction of cancer metastasis. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00766-y.

Paracrine HB-EGF signaling reduce enhanced contractile and energetic state of activated decidual fibroblasts by rebalancing SRF-MRTF-TCF transcriptional axis.

Multiple parallels exist between placentation and cancer dissemination at molecular, cellular, and anatomical levels, presenting placentation as a unique model to mechanistically understand the onset of cancer metastasis. In humans, interaction of placenta and the endometrium results eventually in deep invasion of placental extravillous trophoblasts (EVTs) into the maternal stroma, a process similar to stromal trespass by disseminating carcinoma cells. In anticipation of implantation, endometrial fibroblasts (ESFs) undergo a process called decidualization during the secretory phase of the menstrual cycle. Decidualization, among other substantial changes associated with ESF differentiation, also involves a component of fibroblast activation, and myofibroblast transformation. Here, using traction force microscopy, we show that increased cellular contractility in decidualized ESFs is reversed after interaction with EVTs. We also report here the large changes in energetic state of ESFs upon decidualization, showing increased oxidative phosphorylation, mitochondrial competency and ATP generation, as well as enhanced aerobic glycolysis, presenting mechanical contractility and energetic state as new functional hallmarks for decidualization. These energetic changes accompanying the marked increase in contractile force generation in decidualization were reduced in the presence of EVTs. We also show that increase in decidual contractility and mechanical resistance to invasion is achieved by SRF-MRTF transcriptional activation, achieved via increased phosphorylation of fibroblast-specific myosin light chain 9 (MYL9). EVT induced paracrine secretion of Heparin Binding Epidermal Growth Factor (HBEGF), a potent MAPK activator, which shifts the balance of SRF association away from MRTF based transcription, reducing decidual ESF contractility and mechanical resistance to placental invasion. Our results identify a new axis of intercellular communication in the placental bed modulating stromal force generation and resistance to invasion with concurrent downregulation of cellular energetics. These findings have important implications for implantation related disorders, as well as stromal control of cancer dissemination.

Cardiac ultrastructure inspired matrix induces advanced metabolic and functional maturation of differentiated human cardiomyocytes.

The vast potential of human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) in preclinical models of cardiac pathologies, precision medicine, and drug screening remains to be fully realized because hiPSC-CMs are immature without adult-like characteristics. Here, we present a method to accelerate hiPSC-CM maturation on a substrate, cardiac mimetic matrix (CMM), mimicking adult human heart matrix ligand chemistry, rigidity, and submicron ultrastructure, which synergistically mature hiPSC-CMs rapidly within 30 days. hiPSC-CMs matured on CMM exhibit systemic transcriptomic maturation toward an adult heart state, are aligned with high strain energy, metabolically rely on oxidative phosphorylation and fatty acid oxidation, and display enhanced redox handling capability, efficient calcium handling, and electrophysiological features of ventricular myocytes. Endothelin-1-induced pathological hypertrophy is mitigated on CMM, highlighting the role of a native cardiac microenvironment in withstanding hypertrophy progression. CMM is a convenient model for accelerated development of ventricular myocytes manifesting highly specialized cardiac-specific functions.

Targeting p21Cip1 highly expressing cells in adipose tissue alleviates insulin resistance in obesity.

Insulin resistance is a pathological state often associated with obesity, representing a major risk factor for type 2 diabetes. Limited mechanism-based strategies exist to alleviate insulin resistance. Here, using single-cell transcriptomics, we identify a small, critically important, but previously unexamined cell population, p21Cip1 highly expressing (p21high) cells, which accumulate in adipose tissue with obesity. By leveraging a p21-Cre mouse model, we demonstrate that intermittent clearance of p21high cells can both prevent and alleviate insulin resistance in obese mice. Exclusive inactivation of the NF-κB pathway within p21high cells, without killing them, attenuates insulin resistance. Moreover, fat transplantation experiments establish that p21high cells within fat are sufficient to cause insulin resistance in vivo. Importantly, a senolytic cocktail, dasatinib plus quercetin, eliminates p21high cells in human fat ex vivo and mitigates insulin resistance following xenotransplantation into immuno-deficient mice. Our findings lay the foundation for pursuing the targeting of p21high cells as a new therapy to alleviate insulin resistance.

Research on the influence of heat treatment on residual stress of TC4 alloy produced by laser additive manufacturing based on laser ultrasonic technique.

During the laser additive manufacturing (LAM) process large temperature gradients can lead to high level of residual stress. The residual stress can have irreversible effects such as warping and cracking of parts during and post manufacturing. Heat treatment is an effective method to control and eliminate residual stress. In this paper, the TC4 parts are prepared by laser additive manufacturing, and the influence of heat treatment process on residual stress is researched. Laser ultrasonic technology, as an advanced nondestructive testing method, is applied to measure the residual stress under different heat treatment processes for the first time. The surface wave generated by laser is used to evaluate the residual stress. The results show that laser ultrasonic method can complete the in-situ evaluation of residual stress in additive manufacturing components. The residual stress in TC4 deposited specimen is large, the longitudinal stress is obviously greater than the transverse stress, and the maximum residual stress is about half of the yield strength. The residual stress increases gradually from the upper surface to the bottom layer near the substrate before heat treatment. After heat treatment, the residual stress is reduced to low stress level and a small compressive stress appears. The cooling rate and solution temperature are the main factors affecting the residual stress, and the residual stress increases with the increase of cooling rate and solution temperature. The effect of aging temperature and aging time on residual stress is not obvious. The study serves as useful guidelines for engineers to assessment and regulation of residual stress reasonably in LAM.

Molecularly imprinted polymer solid phase extraction coupled with liquid chromatography-high resolution mass spectrometry for the detection of gonyautoxins 2&3 in seawater.

Paralytic shellfish toxins (PSTs) cause risks to human health through food chains. Understanding the change of PSTs in seawater is critical for predicting the safety of seafood. Most reported methods for the detection of PSTs in microalgae or shellfish are not applicable in seawater because of extremely low concentration and matrix interferences. High resolution mass spectrometry (HRMS), quadrupole exactive orbitrap detects molecular ions accurately, and molecularly imprinted solid-phase extraction (MISPE) is recognized effective to reduce the matrix interference. GTXs 2&3 are two of common marine toxins in PSTs. In this study, a sensitive method consisting MISPE and liquid chromatography LC-HRMS was developed for the detection of GTXs 2&3 with a limit of detection (LOD) of 47.4 ng/L in seawater. With this method, samples obtained from the estuaries of the Shuangtaizi and Daliao Rivers were analyzed, and the results indicated the concentrations were lower than LOD in the area under investigation.

Uniaxial Stretching of Cell-Laden Microfibers for Promoting C2C12 Myoblasts Alignment and Myofibers Formation.

Fiber-shaped cellular constructs have attracted increasing attention in the regeneration of blood vessels, nerve networks, and skeletal myofibers. Nevertheless, the generation of functional fiber-shaped cellular constructs suffers from limited appropriate microfiber-based fabrication approaches and the maintenance of regenerated tissue functions. Herein, we demonstrate a silicone-tube-based coagulant bath free method to fabricate tens of centimeters long cell-laden microfibers using single UV exposure without pretreatment of nozzles or microchannels. By modulating the exposure time, the gelatin methacrylate microfibers with tissue-like microstructures and mechanical properties are obtained. Then, a culture system integrated with a pillar well-array based stretching device is used to apply uniaxial stretching with various strain ratios in situ to cell-laden microfibers in a 60 mm petri dish. Cells with improved spreading, elongation, and alignment are obtained under uniaxial stretching. Moreover, the promotional effects of uniaxial stretching on the differentiation of C2C12 myoblasts, the formation, and contractility of myofibers become more pronounced with increasing strain ratio and achieve saturation level as strain ratio up to ∼35%.

Directed Collective Cell Migration Using Three-Dimensional Bioprinted Micropatterns on Thermoresponsive Surfaces for Myotube Formation.

Directed collective cell migration governs cell orientation during tissue morphogenesis, wound healing, and tumor metastasis. Unfortunately, current methods for initiating collective cell migration, such as scratching, laser ablation, and stencils, either introduce uncontrolled cell-injury, involve multiple fabrication processes, or have utility limited to cells with strong cell-cell junctions. Using three-dimensional (3D) bioprinted gelatin methacryloyl (GelMA) micropatterns on temperature-responsive poly(N-isopropylacrylamide) (PNIPAm) coated interfaces, we demonstrate that directed injury-free collective cell migration could occur in parallel and perpendicular directions. After seeding cells, we created cell-free spaces between two 3D bioprinted GelMA micropatterns by lowering the temperature of PNIPAm interfaces to promote the cell detachment. Unlike conventional collective cell migration methods initiated by stencils, we observed well-organized cell migration in parallel and perpendicular to 3D bioprinted micropatterns as a function of the distance between 3D bioprinted micropatterns. We further established the utility of controlled collective cell migration for directed functional myotube formation using 3D bioprinted fingerprintlike micropatterns as well as iris musclelike concentric circular patterns. Our platform is unique for myoblast alignment and myotube formation because it does not require anisotropic guidance cues. Together, our findings establish how to achieve controlled collective cell migration, even at the macroscale, for tissue engineering and regeneration.

Stress stiffened silicon nitride micro bridges array as substrate with tunable stiffness for cell culture.

Recently, interactions between one-dimensional structural stiffness of physical micro environments and cell biological process have been widely studied. However in previous studies, the influence of structural stiffness on biological process was coupled with the influence of micro fiber curvature. Therefore decoupling the influences of fiber curvature and structural stiffness on cell biological process is of prime importance. In this study, we proposed a novel cell culture substrate comprised of silicon nitride bridges whose structure stiffness can be regulated by altering the axial residual stress without changing material and geometry properties. Both theoretical calculations and finite element simulations were performed to study the influence of residual stress on structure stiffness of bridges. Then multi-positions AFM bending tests were implemented to measure local stiffness of a single micro bridge so as to verify our predictions. NIH/3T3 mouse fibroblast cells were cultured on our substrates to examine the feasibility of the substrate application for investigating cellular response to microenvironment with variable stiffness. The results showed that cells on the edge region near bridge ends were more spread, elongated and better aligned along the bridge axial direction than those on the bridge center region. The results suggest that cells can sense and respond to the differences of stiffness and stiffness gradient between the edge and the center region of the bridges, which makes this kind of substrates can be applied in some biomedical fields, such as cell migration and wound healing.