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Interference reflection microscopy (IRM) is a powerful, label-free technique to visualize the surface structure of biospecimens. However, stray light outside a focal plane obscures the surface fine structures beyond the diffraction limit (dxy ≈ 200 nm). Here, we developed an advanced interferometry approach to visualize the surface fine structure of complex biospecimens, ranging from protein assemblies to single cells. Compared to 2-D, our unique 3-D structure illumination introduced to IRM enabled successful visualization of fine structures and the dynamics of protein crystal growth under lateral (dx-y ≈ 110 nm) and axial (dx-z ≤ 5 nm) resolutions and dynamical adhesion of microtubule fiber networks with lateral resolution (dx-y ≈ 120 nm), 10 times greater than unstructured IRM (dx-y ≈ 1000 nm). Simultaneous reflection/fluorescence imaging provides new physical fingerprints for studying complex biospecimens and biological processes such as myogenic differentiation and highlights the potential use of advanced interferometry to study key nanostructures of complex biospecimens.
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Interferometría , Iluminación , Microscopía de Interferencia/métodos , Microtúbulos , ProteínasRESUMEN
Synthetic protocols providing mechanical patterns to culture substrate are essential to control the self-condensation of cells for organoid engineering. Here, we present a protocol for preparing hydrogels with mechanical patterns. We describe steps for hydrogel synthesis, mechanical evaluation of the substrate, and time-lapse imaging of cell self-organization. This protocol will facilitate the rational design of culture substrates with mechanical patterns for the engineering of various functional organoids. For complete details on the use and execution of this protocol, please refer to Takebe et al. (2015) and Matsuzaki et al. (2014, 2022).1,2,3.
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Hidrogeles , OrganoidesRESUMEN
Focused irradiation with ultrashort laser pulses realized the fine spatiotemporal control of ice crystallization in supercooled water. An effective multiphoton excitation at the laser focus generated shockwaves and bubbles, which acted as an impulse for inducing ice crystal nucleation. The impulse that was localized close to the laser focus and accompanied by a small temperature elevation allowed the precise position control of ice crystallization and its observation with spatiotemporal resolution of micrometers and microseconds using a microscope. To verify the versatility of this laser method, we also applied it using various aqueous systems (e.g., plant extracts). The systematic study of crystallization probability revealed that laser-induced cavitation bubbles play a crucial role in inducing ice crystal nucleation. This method can be used as a tool for studying ice crystallization dynamics in various natural and biological phenomena.
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Bio-orthogonal ligations that crosslink living cells with a substrate or other cells require high stability and rapid kinetics to maintain the nature of target cells. In this study, we report water-soluble cyclooctadiyne (WS-CODY) derivatives that undergo an ion-pair enhanced double-click reaction. The cationic side chain of WS-CODY accelerated the kinetics on the azide-modified cell surface due to proximity effect. Cationic WS-CODY was able to crosslink azide-modified, poorly adherent human lung cancer PC-9 cells not only to azide-grafted glass substrates but also to other cells within 5-30 min. We discovered that cell-substrate crosslinking induced the ITGA5 gene expression, whereas cell-cell crosslinking induced the CTNNA1 gene, according to the adhesion partner. Ion-pair-enhanced WS-CODY can be applied to a wide range of cells with established azide modifications and is expected to provide a powerful tool to regulate cell-substrate and cell-cell interactions.
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Spatially controlled self-organization represents a major challenge for organoid engineering. We have developed a mechanically patterned hydrogel for controlling self-condensation process to generate multi-cellular organoids. We first found that local stiffening with intrinsic mechanical gradient (IG > 0.008) induced single condensates of mesenchymal myoblasts, whereas the local softening led to stochastic aggregation. Besides, we revealed the cellular mechanism of two-step self-condensation: (1) cellular adhesion and migration at the mechanical boundary and (2) cell-cell contraction driven by intercellular actin-myosin networks. Finally, human pluripotent stem cell-derived hepatic progenitors with mesenchymal/endothelial cells (i.e., liver bud organoids) experienced collective migration toward locally stiffened regions generating condensates of the concave to spherical shapes. The underlying mechanism can be explained by force competition of cell-cell and cell-hydrogel biomechanical interactions between stiff and soft regions. These insights will facilitate the rational design of culture substrates inducing symmetry breaking in self-condensation of differentiating progeny toward future organoid engineering.
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Cell-coupled field-effect transistor (FET) biosensors have attracted considerable attention because of their high sensitivity to biomolecules. The use of insect cells (Sf21) as a core sensor element is advantageous due to their stable adhesion to sensors at room temperature. Although visualization of the insect cell-substrate interface leads to logical amplification of signals, the spatiotemporal processes at the interfaces have not yet been elucidated. We quantitatively monitored the adhesion dynamics of Sf21 using interference reflection microscopy (IRM). Specific adhesion signatures with ring-like patches along the cellular periphery were detected. A combination of zeta potential measurements and lectin staining identified specific glycoconjugates with low electrostatic potentials. The ring-like structures were disrupted after cholesterol depletion, suggesting a raft domain along the cell periphery. Our results indicate dynamic and asymmetric cell adhesion is due to low electrostatic repulsion with fluidic sugar rafts. We envision the logical design of cell-sensor interfaces with an electrical model that accounts for actual adhesion interfaces.
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Colesterol , Lectinas , Animales , Adhesión Celular , Glicoconjugados , Insectos , Azúcares , TemperaturaRESUMEN
The stabilization mechanism of the Zn-terminated (Zn-) ZnO(0001) surface in electrolyte solutions has been investigated by using atomic-resolution liquid-environment atomic force microscopy (AFM) and an electrochemical method. The electrochemically measured pH dependence of the flat band potential of the Zn-ZnO(0001) surface indicated the adsorption of OH groups onto the (0001) surface in the wide pH range of 1-13. Atomic-scale AFM images of the Zn-ZnO(0001) surface showed a well-ordered hydroxide superstructure in an alkaline solution but a disordered structure in an acidic solution, which is probably attributed to the rapid diffusion of the adsorbed OH groups. Furthermore, the density of the O-terminated step edge on the Zn-ZnO(0001) surface in an acidic solution was higher than that in an alkaline solution. From these findings, we concluded that the excess positive charges of the Zn-ZnO(0001) surface are compensated by the adsorbed OH groups and the O-terminated step edges. In acidic solutions, a higher density of the O-terminated step edge is required for charge compensation. In addition, it was found that potential-dependent reversible surface reconstruction occurs in the local transition area with disordered step orientation by electrochemical AFM. We concluded that the reconstruction compensates the excess surface charges of the local transition area which are induced and varied by potential-dependent local surface states.
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The fluorescent metabolic labeling of microorganisms genome is an advanced imaging technique to observe and study the native shapes, structural changes, functions, and tracking of nucleic acids in single cells or tissues. We have attempted to visualize the newly synthesized DNA within the intact nucleoid of ice-embedded proliferating cells of Escherichia coli K-12 (thymidine-requiring mutant, strain N4316) via correlative light-electron microscopy. For that purpose, erythrosine-11-dUTP was synthesized and used as a modified analog of the exogenous thymidine substrate for metabolic incorporation into the bacterial chromosome. The formed fluorescent genomic DNA during in cellulo polymerase reaction caused a minimal cellular arrest and cytotoxicity of E. coli at certain controlled conditions. The stained cells were visualized in typical red emission color via an epifluorescence microscope. They were further ice-embedded and examined with a Hilbert differential contrast transmission electron microscopy. At high-resolution, the ultrastructure of tagged nucleoid appeared with significantly higher electron dense in comparison to the unlabeled one. The enhanced contrast areas in the chromosome were ascribed to the presence of iodine contents from erythrosine dye. The presented labeling approach might be a powerful strategy to reveal the structural and dynamic changes in natural DNA replication including the relationship between newly synthesized in vivo nucleic acid and the physiological state of the cell.
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ADN Bacteriano/genética , Nucleótidos de Desoxiuracil/química , Eritrosina/química , Escherichia coli K12/genética , Escherichia coli K12/ultraestructura , Microscopía Electrónica de Transmisión/métodos , Eritrosina/análogos & derivados , Pruebas de Sensibilidad Microbiana , Microscopía Fluorescente/métodos , Microscopía de Contraste de Fase/métodos , Conformación Molecular , Coloración y Etiquetado/métodosRESUMEN
Programmable cell adhesion with DNA hybridization is a promising approach for fabricating various tissue architectures without sophisticated instrumentation. However, little is known about how this artificial interaction influences the binding of cell adhesion proteins, E-cadherin. In this work, we designed a planar and fluid lipid membrane displaying E-cadherin and/or single-strand DNA with well-defined densities. Visualization of cells on membranes by fluorescence and interference microscopy revealed cell adhesion to be a two-step process: artificial adhesion by DNA hybridization within a few minutes followed by biological adhesion via cadherin-cadherin binding within hours. Furthermore, we discovered that DNA hybridization can substantially facilitate E-cadherin-mediated cell adhesion. The promotive effect is probably due to the enforced binding between E-cadherin molecules in geometrical confinement between two membranes. Our in vitro model of cell adhesion can potentially be used to design functional synthetic molecules that can regulate cell adhesion via cell adhesion proteins for tissue engineering.
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We performed molecular dynamics simulations of a lipid bilayer consisting of POPC and cholesterol at temperatures from 283 to 308K and cholesterol concentrations from 0 to 50% mol/mol. The purpose of this study was to look for the existence of structural differences in the region delimited by these parameters and, in particular, in a region where coexistence of liquid disordered and liquid ordered phases has been proposed. Our interest in this range of concentration and temperature responds to the fact that polyene ionophore activity varies considerably along it. Two force fields, CHARMM36 and Slipids, were compared in order to determine the most suitable. Both force fields predict non-monotonic behaviors consistent with the existence of phase transitions. We found the presence of lateral structural heterogeneity, statistical in nature, in some of the bilayers occurring in this range of temperatures and sterol concentrations. This heterogeneity was produced by correlated ordering of the POPC tails and not due to cholesterol enrichment, and lasts for tens of nanoseconds. We relate these observations to the action of polyenes in these membranes.
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Colesterol/química , Membrana Dobles de Lípidos/química , Simulación de Dinámica Molecular , Fosfatidilcolinas/química , Microscopía de Fuerza Atómica , Transición de Fase , TemperaturaRESUMEN
The effect of cholesterol and ergosterol on supported lipid bilayers composed of 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and egg sphingomyelin (eSM) in a 1/1â¯M ratio was studied using atomic force microscopy. The addition of ergosterol or cholesterol to these membranes considerably modifies both the structure and the dynamics of the domains present in them. The height of the eSM enriched domains increases with concentration of both sterols, but more markedly with ergosterol. The height of the POPC enriched domains increases with concentration in a similar manner for both sterols. This effect is larger for eSM than for POPC when ergosterol, not cholesterol, is present. Domain coverage increases with both sterols at 5â¯mol% but decreases at 20â¯mol% and almost disappears at 40â¯mol%. The size of the eSM enriched domains decreases with sterol concentration, more markedly with cholesterol. Bilayer rupture forces show that overall stiffness increases with the addition of 5â¯mol% cholesterol, but only for the eSM enriched domains with ergosterol at the same concentration. At larger sterol concentrations the stiffness of both regions becomes reduced. At 40â¯mol% sterol concentration, both membranes present the same rupture force value. To gain mechanistic insight into these observations we performed Quantum Mechanical calculations and Molecular Dynamics simulations of the sterol molecules. We found that conformational freedom for the sterol molecules is quite different. This difference might be behind the observed phenomena. Finally, the different action of sterols on membrane properties is related to the sterol-dependent ionophoretic activity of polyene antibiotics.
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Colesterol/química , Ergosterol/química , Membrana Dobles de Lípidos/química , Microdominios de Membrana/química , Microdominios de Membrana/ultraestructura , Fosfatidilcolinas/química , Esfingomielinas/química , Liposomas Unilamelares/químicaRESUMEN
The real time monitoring and quantification of the concentration of highly fluorescent nitrogen-doped carbon nanodots (C-dots) in eukaryotic Tobacco bright yellow-2 (BY-2) plant cells was investigated by fluorescence and confocal microscopy. The quantitative measurement of their fluorescent emission intensity was possible because of the high photo-resistance, good water solubility and the absence of fading effect of the nanoparticles, which is frequent occurred problem of the conventional organic dyes. The microscopic analysis revealed that C-dots entered generally into the cells through endocytosis and caused negligible cytotoxicity. The multicolor cellular imaging of labeled Tobacco BY-2 demonstrates that the cells were in good health conditions and any blinking artifacts were not observed. The quantification of fluorescence emission intensity was carried out in the intracellular regions where the relationship between the C-dots concentration and relative emission was linear. Based on a control experiment with fluorescence liposomes with known dependence between C-dots concentration and emission, we were able to determine the amount of accumulated nanoparticles in the inner compartments of the eukaryotic cell through subsequent digital image analysis. The reported microscopic approach may be used for accurate testing and direct examination of the drug internalization mechanisms by C-dots as sensitive probes in single cells or tissues.
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Carbono/metabolismo , Nanopartículas/metabolismo , Nicotiana/metabolismo , Nitrógeno/metabolismo , Transporte Biológico , Carbono/química , Células Cultivadas , Fluorescencia , Microscopía Confocal , Nanopartículas/química , Nitrógeno/química , Nicotiana/químicaRESUMEN
The native shape and intracellular distribution of newly synthesized DNA was visualized by correlative (light and electron) microscopy in ice embedded whole cells of Escherichia coli. For that purpose, the commercially available modified nucleoside triphosphate named BODIPY® FL-14-dUTP was enzymatically incorporated in vivo into the genome of E. coli mutant K12 strain, which cannot synthesize thymine. The successful incorporation of this thymidine analogue was confirmed first by fluorescence microscope, where the cells were stained in the typical for bodipy green color. Later the preselected labeled E. coli were observed by Hilbert Differential Transmission Electron Microscope (HDC TEM) and the distribution of elemental boron (contained in bodipy) was visualized at high-resolution by an electron spectroscopic imaging (ESI) technique. The practical detection limit of boron was found to be around 5 â¼ 10 mmol/kg in area of 0.1 µm2 , which demonstrated that ESI is a suitable approach to study the cytochemistry and location of labeled nucleic fragments within the cytoplasmic chromosomal area. In addition, the fine cellular fibrous and chromosomal ultrastructures were revealed in situ by combing of phase-plate HDC TEM and ESI. The obtained results conclude that the correlation between fluorescent microscopy with phase-plate HDC TEM and ESI is a powerful approach to explore the structural and conformation dynamics of DNA replication machinery in frozen cells close to the living state.
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ADN Bacteriano/química , Escherichia coli/citología , Escherichia coli/genética , Boro/química , Compuestos de Boro , Colorantes Fluorescentes , Hielo , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Manejo de EspecímenesRESUMEN
Physical interactions of four major green tea catechin derivatives with cell membrane models were systemically investigated. Catechins with the galloyl moiety caused the aggregation of small unilamellar vesicles and an increase in the surface pressure of lipid monolayers, while those without did not. Differential scanning calorimetry revealed that, in a low concentration regime (≤10 µM), catechin molecules are not significantly incorporated into the hydrophobic core of lipid membranes as substitutional impurities. Partition coefficient measurements revealed that the galloyl moiety of catechin and the cationic quaternary amine of lipids dominate the catechin-membrane interaction, which can be attributed to the combination of electrostatic and cation-π interactions. Finally, we shed light on the mechanical consequence of catechin-membrane interactions using the Fourier-transformation of the membrane fluctuation. Surprisingly, the incubation of cell-sized vesicles with 1 µM galloyl catechins, which is comparable to the level in human blood plasma after green tea consumption, significantly increased the bending stiffness of the membranes by a factor of more than 60, while those without the galloyl moiety had no detectable influence. Atomic force microscopy and circular dichroism spectroscopy suggest that the membrane stiffening is mainly attributed to the adsorption of galloyl catechin aggregates to the membrane surfaces. These results contribute to our understanding of the physical and thus the generic functions of green tea catechins in therapeutics, such as cancer prevention.
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Catequina/análogos & derivados , Membrana Dobles de Lípidos/química , Adsorción , Rastreo Diferencial de Calorimetría , Catequina/química , Catequina/metabolismo , Dicroismo Circular , Dispersión Dinámica de Luz , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Membrana Dobles de Lípidos/metabolismo , Microscopía de Fuerza AtómicaRESUMEN
We report synchronization of Mercury Beating Heart (MBH) oscillators using the environmental coupling mechanism. This mechanism involves interaction of the oscillators with a common medium/environment such that the oscillators do not interact among themselves. In the present work, we chose a modified MBH system as the common environment. In the absence of coupling, this modified system does not exhibit self sustained oscillations. It was observed that, as a result of the coupling of the MBH oscillators with this common environment, the electrical and the mechanical activities of both the oscillators synchronized simultaneously. Experimental results indicate the emergence of both lag and the complete synchronization in the MBH oscillators. Simulations of the phase oscillators were carried out in order to better understand the experimental observations.
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The main constituent of green tea, (-)-Epigallocatechin-3-O-gallate (EGCG), is known to have cancer-specific chemopreventive effects. In the present work, we investigated how EGCG suppresses cell adhesion by comparing the adhesion of human pancreatic cancer cells (AsPC-1 and BxPC-3) and their counterpart, normal human embryonic pancreas-derived cells (1C3D3), in catechin-containing media using organosilane monolayer templates (OMTs). The purpose of this work is (1) to evaluate the quantitativeness in the measurement of cell adhesion with the OMT and (2) to show how green-tea catechins suppress cell adhesion in a cancer-specific manner. For the first purpose, the adhesion of cancer and normal cells was compared using the OMT. The cell adhesion in different type of catechins such as EGCG, (-)-Epicatechin-3-O-gallate (ECG) and (-)-Epicatechin (EC) was also evaluated. The measurements revealed that the anti-adhesion effect of green-tea catechins is cancer-specific, and the order is EGCGâ«ECG>EC. The results agree well with the data reported to date, showing the quantitativeness of the new method. For the second purpose, the contact area of cells on the OMT was measured by reflection interference contrast microscopy. The cell-OMT contact area of cancer cells decreases with increasing EGCG concentration, whereas that of normal cells remains constant. The results reveal a twofold action of EGCG on cancer cell adhesion-suppressing cell attachment to a candidate adhesion site and decreasing the contact area of the cells-and validates the use of OMT as a tool for screening cancer cell adhesion.
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Catequina/análogos & derivados , Adhesión Celular/efectos de los fármacos , Páncreas/efectos de los fármacos , Neoplasias Pancreáticas/tratamiento farmacológico , Catequina/administración & dosificación , Catequina/química , Línea Celular Tumoral , Humanos , Neoplasias Pancreáticas/patología , Té/químicaRESUMEN
In this contribution, they have attempted to develop a labeling technique for in vivo imaging of functionally active plasmid DNA in cyanobacterial cells through its decoration with semiconductor quantum dots (Qdots) as fluorescent nanoprobes. For that purpose biotinylated plasmid slr2060 DNA was conjugated with Qdots-streptavidine. The intact DNA was visualized in a single green color by light microscopy. These Qdots-DNA conjugates were capable of expressing the acyltransferase enzyme. Qdots-DNA conjugates and confocal microscope imaging technique were adopted to visualize the gene transport across the membrane of the live cyanobacteria cell in real time. Long-term kinetic study enabled to reveal the steps of extracellular and intracellular microenvironment for plasmid transportation into the live cell. To confirm these processes a confocal microscope and indicator plate assay test were applied in tandem. In this contribution, Qdots-labeled plasmid DNA was utilized for the first time for long-term intracellular imaging studies in cyanobacteria species PCC6803. The results showed that the Qdots-labeled plasmid DNA detection could be used as a powerful labeling technique for visualization of exogenous DNA entry and tracking into living cells by confocal microscopy.
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Cianobacterias/genética , ADN Bacteriano/química , Puntos Cuánticos/química , Proteínas Bacterianas/metabolismo , Microscopía ConfocalRESUMEN
Adhesion of cancer cells with different metastatic potential and anticancer drug resistance has been quantitatively evaluated by using self-assembled monolayer (SAM)-patterned substrates and reflection interference contrast microscopy (RICM). Cell-adhesive SAM spots with optimized diameter could prevent cell-cell adhesion and thus allowed the systematic evaluation of statistically reliable numbers of contact area between single cancer cells and substrates by RICM. The statistical image analysis revealed that highly metastatic mouse melanoma cells showed larger contact area than lowly metastatic cells. We also found that both cancer cell types exhibited distinct transition from the "strong" to "weak" adhesion states with increase in the concentration of (-)-epigallocatechin gallate (EGCG), which is known to exhibit cancer preventive activity. Mathematical analysis of the adhesion transition revealed that adhesion of the highly metastatic mouse melanoma cells showed more EGCG tolerance than that of lowly metastatic cells. Moreover, time-lapse RICM observation revealed that EGCG weakened cancer cell adhesion in a stepwise manner, probably via focal adhesion complex. These results clearly indicate that contact area can be used as a quantitative measure for the determination of cancer phenotypes and their drug resistance, which will provide physical insights into the mechanism of cancer metastasis and cancer prevention.
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Anticarcinógenos/farmacología , Catequina/análogos & derivados , Adhesión Celular/efectos de los fármacos , Microscopía de Interferencia/métodos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Animales , Catequina/farmacología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Humanos , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/patología , Ratones , Metástasis de la Neoplasia/tratamiento farmacológico , Metástasis de la Neoplasia/patologíaRESUMEN
We present a new platform to give stochastic mechanical stimuli to cells for their characterization. There nano- and micrometer scaled fluctuations are generated by an engineered motor protein system of kinesin-microtubules (MTs) on a solid surface. Cells have abilities to deform in many ways during homeostatic metabolism, tissue forming processes, cancer developments, and so on. Namely, cells in biological tissues are exposed to noise-like stochastic movements at nano- and micrometer-scales, which mainly come from the mechanical environment surrounding the cells. Although cells seem to have the potential to respond to such types of mechanical stimuli, the influences on cellular behaviors are poorly understood. As a first attempt to verify an effect of noise-like mechanical stimuli in vitro, we prepared a system to give stochastic mechanical stimuli to cells using a technique of in vitro motility assay for a kinesin-MT system. An active substrate was obtained by integrating movements of MTs on a kinesin-coated glass surface via cross-linkage, and stochastic mechanical stimuli at the cell-scale were successfully applied to the seeded cells. There, traveling distances of the cells over one cell length were observed until they started to adhere. When metastatic melanoma cells were exposed to the stochastic mechanical stimuli, unusually long protrusions or extensions of cell bodies were observed. Cellular aggregations were also promoted through the movements on this active substrate which could disturb the landing and enhance the collisions of the cells. This approach giving mechanical stimuli to cells in a stochastic manner at nano- and micrometer-scales might allow us to uncover unknown behaviors of cells, which might contribute to research fields requiring our understanding on the mechanical nature of cells, such as cancer diagnosis and regenerative medicine.
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In healthy joints, hyaline cartilage covering the joint surfaces of bones provides cushioning due to its unique mechanical properties. However, because of its limited regenerative capacity, age- and sports-related injuries to this tissue may lead to degenerative arthropathies, prompting researchers to investigate a variety of cell sources. We recently succeeded in isolating human cartilage progenitor cells from ear elastic cartilage. Human cartilage progenitor cells have high chondrogenic and proliferative potential to form elastic cartilage with long-term tissue maintenance. However, it is unknown whether ear-derived cartilage progenitor cells can be used to reconstruct hyaline cartilage, which has different mechanical and histological properties from elastic cartilage. In our efforts to develop foundational technologies for joint hyaline cartilage repair and reconstruction, we conducted this study to obtain an answer to this question. We created an experimental canine model of knee joint cartilage damage, transplanted ear-derived autologous cartilage progenitor cells. The reconstructed cartilage was rich in proteoglycans and showed unique histological characteristics similar to joint hyaline cartilage. In addition, mechanical properties of the reconstructed tissues were higher than those of ear cartilage and equal to those of joint hyaline cartilage. This study suggested that joint hyaline cartilage was reconstructed from ear-derived cartilage progenitor cells. It also demonstrated that ear-derived cartilage progenitor cells, which can be harvested by a minimally invasive method, would be useful for reconstructing joint hyaline cartilage in patients with degenerative arthropathies.