The biophysical properties of the extracellular matrix (ECM) dictate tissue-specific cell behaviour. In the skeleton system, bone shows the potential to adapt its architecture and contexture to environmental rigidity via the bone remodelling process, which involves chondrocytes, osteoblasts, osteoclasts, osteocytes and even peripheral bone marrow-derived stem/stromal cells (BMSCs). In the current study, we generated stiff (~1 014 ± 56) kPa, Young's modulus) and soft (~46 ± 11) kPa silicon-based elastomer polydimethylsiloxane (PDMS) substrates by mixing curing agent into oligomeric base at 1:5 and 1:45 ratios, respectively, and investigated the influence of substrate stiffness on the cell behaviours by characterizing cell spreading area, cell cytoskeleton and cell adhesion capacity. The results showed that the cell spreading areas of chondrocytes, osteoblasts, osteoclasts, osteocytes and BMSCs were all reduced in the soft substrate relative to those in the stiff substrate. F-actin staining confirmed that the cytoskeleton was also changed in the soft group compared to that in the stiff group. Vinculin in focal adhesion plaques was significantly decreased in response to soft substrate compared to stiff substrate. This study establishes the potential correlation between microenvironmental mechanics and the skeletal system, and the results regarding changes in cell spreading area, cytoskeleton and cell adhesion further indicate the important role of biomechanics in the cell-matrix interaction.
Actins ; Cell Adhesion ; Elastic Modulus ; Focal Adhesions ; physiology ; Humans ; Vinculin ; analysis ; metabolism

PURPOSE: To evaluate adherence of human gingival fibroblasts (HGFs) to transmucosal abutment of dental implant with different surface conditions with time and to investigate the roles of focal adhesion linker proteins (FALPs) involved in HGFs adhesion to abutment surfaces. MATERIALS AND METHODS: Morphologies of cultured HGFs on titanium and ceramic discs with different surface were observed by scanning electron microscopy. Biocompatibility and focal adhesion were evaluated by ultrasonic wave application and cell viability assay. FALPs expression levels were assessed by RT-PCR and western blot. RESULTS: There seemed to be little difference in biocompatibility and adhesion strength of HGFs depending on the surface conditions and materials. In all experimental groups, the number of cells remaining on the disc surface after ultrasonic wave application increased more than 2 times at 3 days after seeding compared to 1-day cultured cells and this continued until 7 days of culture. FALPs expression levels, especially of vinculin and paxillin, also increased in 5-day cultured cells compared to 1-day cultured fibroblasts on the disc surface. CONCLUSION: These results might suggest that the strength of adhesion of fibroblasts to transmucosal abutment surfaces increases with time and it seemed to be related to expressions of FALPs.
Cell Survival ; Cells, Cultured ; Ceramics ; Dental Implants ; Fibroblasts ; Focal Adhesions ; Humans ; Microscopy, Electron, Scanning ; Paxillin ; Proteins ; Seeds ; Titanium ; Ultrasonics ; Vinculin

STATEMENT OF PROBLEM: Mechanisms of tissue-implant interaction and the effect of the implant surface on the behavior of cells has not yet been clarified. PURPOSE: This study was performed to investigate the tissue reaction to the titanium alloy submerged into rat peritoneum in vivo. MATERIALS AND METHODS: Titanium alloys (titanium-13Zirconium-6Niobium) were inserted inside the peritoneal cavity of Sprague Dawley rats. After 3 months, the tissue formed around the inserted titanium alloys were examined with a light-microscope. Tissue reaction around the material was analyzed by confocal microscopy to evaluate their biocompatibility in a living body. RESULTS: In in vivo study, foreign body type multinucleated giant cells were found in the fibrous tissue formed as a reaction to the foreign material (4 in 20 cases), but the inflammatory reaction was very weak. After experiment, the contaminants of biomaterials was removed from living tissue. In confocal microscopy, we observed that the staining of vinculin and actin showed mixed appearance. In a few cases, we found that the staining of vinculin and beta-catenin showed the prominent appearance. CONCLUSION: We found that titanium-13Zirconium-6Niobium alloy was an excellent biomaterial.
Actins ; Alloys* ; Animals ; beta Catenin ; Biocompatible Materials ; Foreign Bodies ; Giant Cells ; Microscopy, Confocal ; Peritoneal Cavity ; Peritoneum ; Rats ; Rats, Sprague-Dawley ; Titanium* ; Vinculin

The importance of soft tissue response to implant abutments has become one of the major issues in current implant dentistry. To date, numerous studies have emphasized on maintaining connective tissue barriers in quantity, as well as in quality for the long term success of dental implants. The cells mainly consisting the soft tissue around dental implants are fibroblasts and epithelial cells. The mechanism of the fibroblasts'adhesions to certain substrata can be explained by the 'focal adhesion'theory. On the other hand, epithelial cells adhere to the substratum via hemidesmosomes. The typical integrin-mediated adhesions of cells to certain matrix are called 'cell-matrix adhsions'. The focal adhesion complex of fibroblasts, in relation to the cell-matrix adhsions, consists of the extracellular matrix(ECM) such as fibronectin, the transmembrane proteins such as integrins, the intracellular cytoplasmic proteins such as vinculin, talin, and more, and the cytoskeletal structures such as filamentous actin and microtubules. The mechanosensory function of integrins and focal adhesion complexes are considered to play a major role in the cells'adhesion, migration, proliferation, differentiation, division, and even apoptosis. The '3-D matrix adhesions'defined by Cukierman et al. makes a promising future for the verification of the actual process of the cell-matrix adhesions in vivo and can be applied to the field of implant dentistry in relation to obtaining strong soft tissue attachment to the implant abutments.
Actins ; Apoptosis ; Cell-Matrix Junctions* ; Connective Tissue ; Cytoplasm ; Dental Implants* ; Dentistry ; Epithelial Cells ; Fibroblasts ; Fibronectins ; Focal Adhesions ; Hand ; Hemidesmosomes ; Integrins ; Microtubules ; Talin ; Vinculin

The importance of soft tissue response to implant abutments has become one of the major issues in current implant dentistry. To date, numerous studies have emphasized on maintaining connective tissue barriers in quantity, as well as in quality for the long term success of dental implants. The cells mainly consisting the soft tissue around dental implants are fibroblasts and epithelial cells. The mechanism of the fibroblasts'adhesions to certain substrata can be explained by the 'focal adhesion'theory. On the other hand, epithelial cells adhere to the substratum via hemidesmosomes. The typical integrin-mediated adhesions of cells to certain matrix are called 'cell-matrix adhsions'. The focal adhesion complex of fibroblasts, in relation to the cell-matrix adhsions, consists of the extracellular matrix(ECM) such as fibronectin, the transmembrane proteins such as integrins, the intracellular cytoplasmic proteins such as vinculin, talin, and more, and the cytoskeletal structures such as filamentous actin and microtubules. The mechanosensory function of integrins and focal adhesion complexes are considered to play a major role in the cells'adhesion, migration, proliferation, differentiation, division, and even apoptosis. The '3-D matrix adhesions'defined by Cukierman et al. makes a promising future for the verification of the actual process of the cell-matrix adhesions in vivo and can be applied to the field of implant dentistry in relation to obtaining strong soft tissue attachment to the implant abutments.
Actins ; Apoptosis ; Cell-Matrix Junctions* ; Connective Tissue ; Cytoplasm ; Dental Implants* ; Dentistry ; Epithelial Cells ; Fibroblasts ; Fibronectins ; Focal Adhesions ; Hand ; Hemidesmosomes ; Integrins ; Microtubules ; Talin ; Vinculin

OBJECTIVETo investigate the expressions of vinculin (VCL) and the androgen receptor (AR) in benign prostatic hyperplasia (BPH) and prostate cancer (PCa) and analyze their relationship with the clinical stage and pathological grade of PCa and the level of PSA.

METHODSWe detected the expressions of VCL and AR in 18 cases of BPH and 38 cases of PCa by immunohistochemistry, analyzed the differences of VCL and AR expressions in BPH and PCa in different clinical stages and pathological grades of PCa, compared the primary levels of PSA, and studied their correlations.

RESULTSThe positive rate of VCL was significantly higher in PCa than in BPH tissues (P < 0.05), while that of AR showed no significant differences between the two groups (P > 0.05). Both the expressions of VCL and AR were closely related with the clinical stage and pathological grade of PCa (P < 0.05), but not with the PSA level (P > 0.05). There was a positive correlation between the expressions of VCL and AR in PCa tissues (r = 0.489, P < 0.05).

CONCLUSIONVCL is expressed differently in BPH and PCa, which may serve as an indicator for the differential diagnosis of benign and malignant prostate diseases. The expressions of AR and VCL are gradually reduced with the progression of PCa, with a positive correlation between them, and could be used jointly to evaluate the progression and prognosis of PCa.

Aged ; Aged, 80 and over ; Humans ; Male ; Middle Aged ; Prostatic Hyperplasia ; metabolism ; pathology ; Prostatic Neoplasms ; metabolism ; pathology ; Receptors, Androgen ; metabolism ; Vinculin ; metabolism

BACKGROUND: The mechanical deformability of cancer cells has attracted particular attention as an emerging biomarker for the prediction of anti-cancer drug sensitivity. Nevertheless, it has not been possible to establish a general rubric for the identification of drug susceptibility in breast cancer cells from a mechanical perspective. In the present study, we investigated the mechanical alteration associated with resistance to adjuvant therapy in breast cancer cells. METHODS: We performed an ‘atomic force microscopy (AFM)-based nanomechanical study’ on ‘drug-sensitive (MCF-7)’ and ‘drug-resistant (MCF-7/ADR)’ breast cancer cells. We also conducted cell viability tests to evaluate the difference in doxorubicin responsiveness between two breast cancer cell lines. We carried out a wound closure experiment to investigate the motility changes associated with chemotherapeutic resistance. To elucidate the changes in molecular alteration that accompany chemotherapeutic resistance, we investigated the expression of vinculin and integrin-linked kinase-1–which are proteins involved in substrate adhesion and the actin cytoskeleton–using Western blotting analysis. RESULTS: A MTT assay confirmed that the dose-dependent efficacy of doxorubicin was reduced in MCF-7/ADR cells compared to that in MCF-7 cells. The wound assay revealed enhanced two-dimensional motility in the MCF-7/ADR cells. The AFM mechanical assay showed evidence that the drug-resistant breast cancer cells exhibited a significant decrease in mechanical deformability compared to their drug-sensitive counterparts. The mechanical alteration in the MCF-7/ADR cells was accompanied by upregulated vinculin expression. CONCLUSIONS: The obtained results manifestly showed that the altered mechanical signatures–including mechanical deformability and motility–were closely related with drug resistance in the breast cancer cells. We believe that this investigation has improved our understanding of the chemotherapeutic susceptibility of breast cancer cells.
Actins ; Biophysics ; Blotting, Western ; Breast Neoplasms ; Breast ; Cell Line ; Cell Survival ; Doxorubicin ; Drug Resistance ; Drug Resistance, Multiple ; Elastic Modulus ; MCF-7 Cells ; Microscopy, Atomic Force ; Vinculin ; Wounds and Injuries

Objective: To investigate the effect of adenovirus-mediated shRNA down-regulating phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression on vinculin, filamin A, and cortactin in activated hepatic stellate cells (HSCs). Methods: Activated rats hepatic stellate cell line (HSC-T6) was cultured in vitro. Recombinant adenovirus Ad-shRNA/PTEN carrying PTEN targeted RNA interference sequence [short hairpin RNA (shRNA)] and empty control virus Ad-GFP were transfected into HSCs. The PTEN mRNA and protein expression of HSCs in each group were detected by real-time fluorescence quantitative PCR and Western blot. The expressional change of vinculin, filamin A and cortactin in HSCs of each group were detected by confocal laser scanning immunofluorescence microscope. Image-pro plus 6.0 software was used for image analysis and processing. The integrated optical density (IOD) of the fluorescence protein expression was measured. The experiment was divided into three groups: control group (DMEM instead of adenovirus solution in the adenovirus transfection step), Ad-GFP group (transfected with empty virus Ad-GFP only expressing green fluorescent protein), and Ad-shRNA/PTEN group (recombinant adenovirus Ad-shRNA/PTEN carrying shRNA targeting PTEN and expressing green fluorescent protein). One-way analysis of variance was used for comparison of mean value among the three groups, and LSD-test was used for comparison between the groups. Results: shRNA targeted PTEN was successfully transfected and the expression of PTEN mRNA and protein in HSC (P < 0.05) was significantly down-regulated. HSCs vinculin was mainly expressed in the cytoplasm. HSCs vinculin fluorescence IOD in the Ad-shRNA/PTEN group (19 758.83 ± 1 520.60) was higher than control (7 737.16 ± 279.93) and Ad-GFP group (7 725.50 ± 373.03) (P < 0.05), but there was no statistically significant difference between control group and Ad-GFP group (P > 0.05). There was no statistically significant difference in the fluorescence IOD of Filamin A among the three groups (P > 0.05), but the subcellular distribution of Filamin A among the three groups were changed. Filamin A in the Ad-shrNA /PTEN HSC group was mainly distributed in the cytoplasm. Filamin A HSC was mainly located in the nucleus.The filamin A HSC in the control group and Ad-GFP group was mainly located in the nucleus. The nucleocytoplasmic ratio of Filamin A in the AD-shrNA /PTEN group (0.60 ± 0.15) was significantly lower than control group (1.20 ± 0.15) and Ad-GFP group (1.08 ± 0.23), P < 0.05. but there was no statistically significant difference in filamin A nucleocytoplasmic ratio of HSC between the control group and the Ad-GFP group (P > 0.05). Cortactin HSCs in the three groups was mainly distributed in the cytoplasm. The cortactin fluorescence IOD of HSCs in the Ad-shRNA/PTEN group was significantly higher than control group (22 959.94 ± 1 710.42) and the Ad-GFP group (22 547.11 ± 1 588.72 ) (P < 0.05), while there was no statistically significant difference in the IOD of cortactin fluorescence in HSCs between the control group and the Ad-GFP group (P > 0.05). Conclusion: The down-regulation of PTEN expression raises the expression of microfilament-binding protein vinculin and cortactin, and changes the subcellular distribution of another microfilament binding protein filamin A, that is, translocation from nucleus to the cytoplasm in activated HSC in vitro.
Adenoviridae/metabolism* ; Animals ; Carrier Proteins ; Cell Proliferation ; Cortactin ; Filamins/genetics* ; Hepatic Stellate Cells/metabolism* ; PTEN Phosphohydrolase/metabolism* ; RNA, Small Interfering/genetics* ; Rats ; Vinculin/genetics*

PURPOSE: The aim of this study is to determine whether certain biomaterials have the potential to support cell attachment. After seeding bone marrow stromal cells onto the biomaterials, we investigated their responses to each material in vitro. METHODS: Rat bone marrow derived stromal cells were used. The biomaterials were deproteinized bovine bone mineral (DBBM), DBBM coated with fibronectin (FN), synthetic hydroxyapatite (HA), HA coated with FN, HA coated with beta-tricalcium phosphate (TCP), and pure beta-TCP. With confocal laser scanning microscopy, actin filaments and vinculin were observed after 6, 12, and 24 hours of cell seeding. The morphological features of cells on each biomaterial were observed using scanning electron microscopy at day 1 and 7. RESULTS: The cells on HA/FN and HA spread widely and showed better defined actin cytoskeletons than those on the other biomaterials. At the initial phase, FN seemed to have a favorable effect on cell adhesion. In DBBM, very few cells adhered to the surface. CONCLUSIONS: Within the limitations of this study, we can conclude that in contrast with DBBM not supporting cell attachment, HA provided a more favorable environment with respect to cell attachment.
Actin Cytoskeleton ; Animals ; Biocompatible Materials ; Bone Marrow ; Bone Substitutes ; Calcium Phosphates ; Cell Adhesion ; Durapatite ; Fibronectins ; Mesenchymal Stromal Cells ; Microscopy, Confocal ; Microscopy, Electron, Scanning ; Rats ; Seeds ; Stem Cells ; Stromal Cells ; Transplants ; Vinculin

OBJECTIVETo explore the molecular mechanism in the formation of unstable plaques.

METHODSThe cDNA microarray E-MTAB-2055 was downloaded from ArrayExpress database to screen the differentially expressed genes in 24 ruptured plaques against 24 stable plaques. Functional enrichment analysis was conducted to define the biological processes and pathways involved in disease progression. The protein-protein interaction network was constructed to identify the risk modules with close interactions. Five pairs of carotid specimens were used to validate 3 differentially expressed genes of the risk modules by real-time PCR.

RESULTSA total of 439 genes showed differential expression in our analysis, including 232 up-regulated and 207 down-regulated genes according to the data filter criteria. Immune-related biological processes and pathways were greatly enriched. The protein-protein interaction network and module analysis suggested that TYROBP, VCL and CXCR4 might play critical roles in the development of unstable plaques, and differential expressions of CXCR4 and TYROBP in carotid plaques were confirmed by real-time PCR.

CONCLUSIONOur study shows the differential gene expression profile, potential biological processes and signaling pathways involved in the process of plaque rupture. TYROBP may be a new candidate disease gene in the pathogenesis of unstable plaques.

Adaptor Proteins, Signal Transducing ; genetics ; Disease Progression ; Down-Regulation ; Gene Expression Profiling ; Humans ; Membrane Proteins ; genetics ; Oligonucleotide Array Sequence Analysis ; Plaque, Atherosclerotic ; genetics ; Protein Interaction Maps ; Real-Time Polymerase Chain Reaction ; Receptors, CXCR4 ; genetics ; Transcriptome ; Up-Regulation ; Vinculin ; genetics