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Biomolecules Apr 2022The increasing demand for bone implants with improved osseointegration properties has prompted researchers to develop various coating types for metal implants. Atomic...
BACKGROUND
The increasing demand for bone implants with improved osseointegration properties has prompted researchers to develop various coating types for metal implants. Atomic layer deposition (ALD) is a method for producing nanoscale coatings conformally on complex three-dimensional surfaces. We have prepared hydroxyapatite (HA) coating on titanium (Ti) substrate with the ALD method and analyzed the biocompatibility of this coating in terms of cell adhesion and viability.
METHODS
HA coatings were prepared on Ti substrates by depositing CaCO films by ALD and converting them to HA by wet treatment in dilute phosphate solution. MC3T3-E1 preosteoblasts were cultured on ALD-HA, glass slides and bovine bone slices. ALD-HA and glass slides were either coated or non-coated with fibronectin. After 48h culture, cells were imaged with scanning electron microscopy (SEM) and analyzed by vinculin antibody staining for focal adhesion localization. An 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) test was performed to study cell viability.
RESULTS
Vinculin staining revealed similar focal adhesion-like structures on ALD-HA as on glass slides and bone, albeit on ALD-HA and bone the structures were thinner compared to glass slides. This might be due to thin and broad focal adhesions on complex three-dimensional surfaces of ALD-HA and bone. The MTT test showed comparable cell viability on ALD-HA, glass slides and bone.
CONCLUSION
ALD-HA coating was shown to be biocompatible in regard to cell adhesion and viability. This leads to new opportunities in developing improved implant coatings for better osseointegration and implant survival.
Topics: Animals; Cattle; Durapatite; Osseointegration; Osteoblasts; Titanium; Vinculin
PubMed: 35625580
DOI: 10.3390/biom12050654 -
Materials (Basel, Switzerland) Dec 2022The adhesion of zirconia and soft tissue is very important for the success of zirconia implants. The aim of this study was to characterize the effects of excimer laser...
The adhesion of zirconia and soft tissue is very important for the success of zirconia implants. The aim of this study was to characterize the effects of excimer laser treatment of zirconia on the adhesion of L929 fibroblasts. In this study, polished zirconia disks treated with an excimer laser were the experimental group and untreated zirconia disks were the control group. Surface roughness and contact angles of zirconia disks were measured. mRNA expression levels of integrin β1 and collagen type I α1 in L929 fibroblasts cultured on zirconia disks were measured using qRT-PCR. Cell morphology was evaluated using 3D laser microscopy and the expression of vinculin was characterized using confocal microscopy. There was no significant difference in the surface roughness of zirconia disks, but contact angles were significantly lower. mRNA expression of integrin β1 was significantly higher at 3, 6 and 24 h and of collagen type I α1 was significantly higher at 6 and 24 h. L929 fibroblasts tended to form elongated microspikes and vinculin colocalization in those microspikes. Furthermore, vinculin was strongly expressed in filopodia of L929 fibroblasts at 24 h. These results suggest that excimer laser treatment improves adhesion between zirconia disks and L929 fibroblasts.
PubMed: 36614454
DOI: 10.3390/ma16010115 -
Nature Communications Dec 2023The ability of cells and tissues to respond differentially to mechanical forces applied in distinct directions is mediated by the ability of load-bearing proteins to...
The ability of cells and tissues to respond differentially to mechanical forces applied in distinct directions is mediated by the ability of load-bearing proteins to preferentially maintain physical linkages in certain directions. However, the molecular basis and biological consequences of directional force-sensitive binding remain unclear. Vinculin (Vcn) is a load-bearing linker protein that exhibits directional catch bonding due to interactions between the Vcn tail domain (Vt) and filamentous (F)-actin. We developed a computational approach to predict Vcn residues involved in directional catch bonding and produced a set of associated Vcn variants with unaltered Vt structure, actin binding, or phospholipid interactions. Incorporation of the variants did not affect Vcn activation but reduced Vcn loading and altered exchange dynamics, consistent with the loss of directional catch bonding. Expression of Vcn variants perturbed the coordination of subcellular structures and cell migration, establishing key cellular functions for Vcn directional catch bonding.
Topics: Actins; Vinculin; Actin Cytoskeleton; Cell Movement; Protein Binding
PubMed: 38097542
DOI: 10.1038/s41467-023-43779-x -
Scientific Reports Oct 2019Adherens Junctions (AJs) are cell-cell adhesion complexes that sense and propagate mechanical forces by coupling cadherins to the actin cytoskeleton via β-catenin and...
Adherens Junctions (AJs) are cell-cell adhesion complexes that sense and propagate mechanical forces by coupling cadherins to the actin cytoskeleton via β-catenin and the F-actin binding protein αE-catenin. When subjected to mechanical force, the cadherin•catenin complex can tightly link to F-actin through αE-catenin, and also recruits the F-actin-binding protein vinculin. In this study, labeling of native cysteines combined with mass spectrometry revealed conformational changes in αE-catenin upon binding to the E-cadherin•β-catenin complex, vinculin and F-actin. A method to apply physiologically meaningful forces in solution revealed force-induced conformational changes in αE-catenin when bound to F-actin. Comparisons of wild-type αE-catenin and a mutant with enhanced vinculin affinity using cysteine labeling and isothermal titration calorimetry provide evidence for allosteric coupling of the N-terminal β-catenin-binding and the middle (M) vinculin-binding domain of αE-catenin. Cysteine labeling also revealed possible crosstalk between the actin-binding domain and the rest of the protein. The data provide insight into how binding partners and mechanical stress can regulate the conformation of full-length αE-catenin, and identify the M domain as a key transmitter of conformational changes.
Topics: Actins; Allosteric Regulation; Animals; Biomechanical Phenomena; Cysteine; Mice; Models, Molecular; Mutant Proteins; Protein Binding; Protein Conformation; Solutions; Sulfhydryl Compounds; Vinculin; alpha Catenin; beta Catenin
PubMed: 31653927
DOI: 10.1038/s41598-019-51816-3 -
Redox Biology Sep 2020Normal high-density lipoprotein (nHDL) in normal, healthy subjects is able to promote angiogenesis, but the mechanism remains incompletely understood. HDL from patients...
Normal high-density lipoprotein (nHDL) in normal, healthy subjects is able to promote angiogenesis, but the mechanism remains incompletely understood. HDL from patients with coronary artery disease may undergo a variety of oxidative modifications, rendering it dysfunctional; whether the angiogenic effect is mitigated by such dysfunctional HDL (dHDL) is unknown. We hypothesized that dHDL compromises angiogenesis. The angiogenic effects of nHDL and dHDL were assessed using endothelial cell culture, endothelial sprouts from cardiac tissue from C57BL/6 mice, zebrafish model for vascular growth and a model of impaired vascular growth in hypercholesterolemic low-density lipoprotein receptor null(LDLr)mice. MiRNA microarray and proteomic analyses were used to determine the mechanisms. Lipid hydroperoxides were greater in dHDL than in nHDL. While nHDL stimulated angiogenesis, dHDL attenuated these responses. Protein and miRNA profiles in endothelial cells differed between nHDL and dHDL treatments. Moreover, nHDL suppressed miR-24-3p expression to increase vinculin expression resulting in nitric oxide (NO) production, whereas dHDL delivered miR-24-3p to inhibit vinculin expression leading to superoxide anion (O) generation via scavenger receptor class B type 1. Vinculin was required for endothelial nitric oxide synthase (eNOS) expression and activation and modulated the PI3K/AKT/eNOS and ERK1/2 signaling pathways to regulate nHDL- and VEGF-induced angiogenesis. Vinculin overexpression or miR-24-3p inhibition reversed dHDL-impaired angiogenesis. The expressions of vinculin and eNOS and angiogenesis were decreased, but the expression of miR-24-3p and lipid hydroperoxides in HDL were increased in the ischemic lower limbs of hypercholesterolemic LDLr mice. Overexpression of vinculin or miR-24-3p antagomir restored the impaired-angiogenesis in ischemic hypercholesterolemic LDLr mice. Collectively, nHDL stimulated vinculin and eNOS expression to increase NO production by suppressing miR-24-3p to induce angiogenesis, whereas dHDL inhibited vinculin and eNOS expression to enhance O generation by delivering miR-24-3p to impair angiogenesis, and that vinculin and miR-24-3p may be therapeutic targets for dHDL-impaired angiogenesis.
Topics: Animals; Coronary Artery Disease; Endothelial Cells; Healthy Volunteers; Humans; Lipoproteins, HDL; Mice; Mice, Inbred C57BL; MicroRNAs; Neovascularization, Physiologic; Nitric Oxide Synthase Type III; Phosphatidylinositol 3-Kinases; Proteomics; Zebrafish
PubMed: 32863238
DOI: 10.1016/j.redox.2020.101642 -
Nature Communications Dec 2015The link between extracellular-matrix-bound integrins and intracellular F-actin is essential for cell spreading and migration. Here, we demonstrate how the actin-binding...
The link between extracellular-matrix-bound integrins and intracellular F-actin is essential for cell spreading and migration. Here, we demonstrate how the actin-binding proteins talin and vinculin cooperate to provide this link. By expressing structure-based talin mutants in talin null cells, we show that while the C-terminal actin-binding site (ABS3) in talin is required for adhesion complex assembly, the central ABS2 is essential for focal adhesion (FA) maturation. Thus, although ABS2 mutants support cell spreading, the cells lack FAs, fail to polarize and exert reduced force on the surrounding matrix. ABS2 is inhibited by the preceding mechanosensitive vinculin-binding R3 domain, and deletion of R2R3 or expression of constitutively active vinculin generates stable force-independent FAs, although cell polarity is compromised. Our data suggest a model whereby force acting on integrin-talin complexes via ABS3 promotes R3 unfolding and vinculin binding, activating ABS2 and locking talin into an actin-binding configuration that stabilizes FAs.
Topics: Actin Cytoskeleton; Actins; Actomyosin; Animals; Cell Polarity; Focal Adhesions; Mice; NIH 3T3 Cells; Protein Binding; Protein Structure, Tertiary; Talin; Vinculin
PubMed: 26634421
DOI: 10.1038/ncomms10038 -
Materials Today. Bio Dec 2022With the adoption of 3-dimensional (3D) cell culture for modelling of cardiac function and regenerative medicine applications, there is an increased need to understand...
With the adoption of 3-dimensional (3D) cell culture for modelling of cardiac function and regenerative medicine applications, there is an increased need to understand cardiomyocyte mechanosensation in 3D. With existing studies of cardiomyocyte mechanosensation primarily focussed on the behaviour of individual cells in a 2-Dimensional context, it is unclear whether mechanosensation is the same in a 3D, multicellular context. In this study, H9C2 cardiac-derived myoblasts were encapsulated as individual cells and as cell spheroids within stiffness gradient gelatin methacryloyl (GelMA) hydrogels to investigate individual and collective cardiac cell mechanosensation in 3D. Over a 3.68-17.52 kPa stiffness range, it was found that H9C2 cells have a limited capacity to adapt their volume to increasing substrate stiffness, demonstrated by the lack of changes in cell volume and shape across the stiffness gradient. Morphological trends were reflected by the expression of the mechanomarkers YAP, MRTF-A and Lamin-A, which were better correlated with cell and nuclear volume than with substrate stiffness. The localisation of YAP and MRTF-A were dependent on the relative volumes of the cytoplasm and nucleus while Lamin-A expression was elevated with increasing cytoplasmic and nuclear volumes. When cultured as spheroids rather than as individual cells, H9C2 cells adopted a distinct morphology with comparably smaller nuclei than individually cultured cells, while retaining the same overall cell volume. As spheroids, H9C2 cells were sensitive to stiffness cues, shown by decreasing YAP and MRTF-A nuclear localisation, increasing Lamin-A expression, and increasing vinculin expression with increasing substrate stiffness. Like the individually cultured H9C2 cells, mechanomarker expression was correlated to volume adaptation. With increasing cytoplasmic volume, YAP and MRTF-A became less nuclear localised, vinculin expression was increased, and with increasing nuclear volume, the Lamin-A expression fincreased. Together, these data suggest that cardiac cell volume adaptation may be enhanced by cell-cell interactions.
PubMed: 36042852
DOI: 10.1016/j.mtbio.2022.100391 -
Cell Reports Apr 2022Blood vessel morphogenesis is driven by coordinated endothelial cell behaviors. Active remodeling of cell-cell junctions promotes cellular plasticity while preserving...
Blood vessel morphogenesis is driven by coordinated endothelial cell behaviors. Active remodeling of cell-cell junctions promotes cellular plasticity while preserving vascular integrity. Here, we analyze the dynamics of endothelial adherens junctions during lumen formation in angiogenic sprouts in vivo. Live imaging in zebrafish reveals that lumen expansion is accompanied by the formation of transient finger-shaped junctions. Junctional fingers are positively regulated by blood pressure, whereas flow inhibition prevents their formation. Using fluorescent reporters, we show that junctional fingers contain the mechanotransduction protein vinculin. Furthermore, genetic deletion of vinculin prevents finger formation, a junctional defect that could be rescued by transient endothelial expression of vinculin. Our findings suggest a mechanism whereby lumen expansion leads to an increase in junctional tension, triggering recruitment of vinculin and formation of junctional fingers. We propose that endothelial cells employ force-dependent junctional remodeling to counteract external forces in order to maintain vascular integrity during sprouting angiogenesis.
Topics: Adherens Junctions; Animals; Cadherins; Endothelial Cells; Intercellular Junctions; Mechanotransduction, Cellular; Neovascularization, Physiologic; Vinculin; Zebrafish; Zebrafish Proteins
PubMed: 35417696
DOI: 10.1016/j.celrep.2022.110658 -
Oncotarget Oct 2017Prostate cancer (PCa) is one of the most common diseases for male population, and the effective treatment for metastatic castration-resistant PCa is still lacking. To...
Prostate cancer (PCa) is one of the most common diseases for male population, and the effective treatment for metastatic castration-resistant PCa is still lacking. To unravel the underlying mechanism of PCa cell migration, we plan to analyze the related crucial proteins and their roles. In our study, we firstly identify the differentially expressed proteins using quantitative proteomics, and confirm their mRNA expression using quantitative polymerase chain reaction (qPCR). The alterations of these proteins at DNA and mRNA levels are obtained from cBioPortal database. Furthermore, the functions of these proteins are evaluated using wound-healing assay. The quantitative proteomics identified vinculin (VCL) and filamin-C (FLNC) as two highly expressed proteins in PC3 cells, and the DNA and mRNA of these two proteins were amplified and upregulated in a part of PCa patients. Knockdown of VCL and FLNC gene expression significantly inhibit PCa cell migration. These findings suggest that VCL and FLNC identified by quantitative proteomics are highly expressed in PCa cells with high migration potential, and they could be effective targets for repressing PCa cell migration, paving a new avenue for the prognosis and treatment of advanced PCa.
PubMed: 29137275
DOI: 10.18632/oncotarget.19397 -
Nature Communications Jan 2017Circadian clocks drive ∼24 h rhythms in tissue physiology. They rely on transcriptional/translational feedback loops driven by interacting networks of clock...
Circadian clocks drive ∼24 h rhythms in tissue physiology. They rely on transcriptional/translational feedback loops driven by interacting networks of clock complexes. However, little is known about how cell-intrinsic circadian clocks sense and respond to their microenvironment. Here, we reveal that the breast epithelial clock is regulated by the mechano-chemical stiffness of the cellular microenvironment in primary cell culture. Moreover, the mammary clock is controlled by the periductal extracellular matrix in vivo, which contributes to a dampened circadian rhythm during ageing. Mechanistically, the tension sensing cell-matrix adhesion molecule, vinculin, and the Rho/ROCK pathway, which transduces signals provided by extracellular stiffness into cells, regulate the activity of the core circadian clock complex. We also show that genetic perturbation, or age-associated disruption of self-sustained clocks, compromises the self-renewal capacity of mammary epithelia. Thus, circadian clocks are mechano-sensitive, providing a potential mechanism to explain how ageing influences their amplitude and function.
Topics: Aging; Amides; Animals; Breast; Breast Diseases; CLOCK Proteins; Cell Self Renewal; Cellular Microenvironment; Circadian Clocks; Circadian Rhythm; Epithelial Cells; Epithelium; Extracellular Matrix; Female; HEK293 Cells; Humans; Mice; Mice, Inbred C57BL; Mice, Transgenic; Primary Cell Culture; Pyridines; RNA, Small Interfering; Signal Transduction; Spheroids, Cellular; Tissue Culture Techniques; Vinculin; rho-Associated Kinases
PubMed: 28134247
DOI: 10.1038/ncomms14287