scholarly journals Periodic Mechanical Stress INDUCES Chondrocyte Proliferation and Matrix Synthesis via CaMKII-Mediated Pyk2 Signaling

2017 ◽  
Vol 42 (1) ◽  
pp. 383-396 ◽  
Author(s):  
Wenwei Liang ◽  
Zeng Li ◽  
Zhen Wang ◽  
Jinchun Zhou ◽  
Huanghe Song ◽  
...  
Keyword(s):  
Mechanical Stress ◽  
Cell Counting ◽  
Synthesis Methods ◽  
Matrix Synthesis ◽  
Gene Expressions ◽  
Chondrocyte Proliferation ◽  
Direct Cell ◽  
Engineered Cartilage ◽  
Signal Activation

Background/Aims: Periodic mechanical stress can promote chondrocyte proliferation and matrix synthesis to improve the quality of tissue-engineered cartilage. Although the integrin β1–ERK1/2 signal cascade has been implicated in periodic mechanical stress-induced mitogenic effects in chondrocytes, the precise mechanisms have not been fully established. The current study was designed to probe the roles of CaMKII and Pyk2 signaling in periodic mechanical stress-mediated chondrocyte proliferation and matrix synthesis. Methods: Chondrocytes were subjected to periodic mechanical stress, proliferation was assessed by direct cell counting and CCK-8 assay; gene expressions were analyzed using quantitative real-time PCR, protein abundance by Western blotting. Results: Mechanical stress, markedly enhanced the phosphorylation levels of Pyk2 at Tyr402 and CaMKII at Thr286. Both suppression of Pyk2 with Pyk2 inhibitor PF431396 or Pyk2 shRNA and suppression of CaMKII with CaMKII inhibitor KN-93 or CaMKII shRNA blocked periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. Additionally, either pretreatment with KN-93 or shRNA targeted to CaMKII prevented the activation of ERK1/2 and Pyk2 under conditions of periodic mechanical stress. Interestingly, in relation to periodic mechanical stress, in the context of Pyk2 inhibition with PF431396 or its targeted shRNA, only the phosphorylation levels of ERK1/2 were abrogated, while CaMKII signal activation was not affected. Moreover, the phosphorylation levels of CaMKII- Thr286 and Pyk2- Tyr402 were abolished after pretreatment with blocking antibody against integrinβ1 exposed to periodic mechanical stress. Conclusion: Our results collectively indicate that periodic mechanical stress promotes chondrocyte proliferation and matrix synthesis through the integrinβ1–CaMKII–Pyk2–ERK1/2 signaling cascade.

scholarly journals Integrin β1 Gene Therapy Enhances in Vitro Creation of Tissue-Engineered Cartilage Under Periodic Mechanical Stress

2015 ◽  
Vol 37 (4) ◽  
pp. 1301-1314 ◽  
Author(s):  
Wenwei Liang ◽  
Chunhui Zhu ◽  
Feng Liu ◽  
Weiding Cui ◽  
Qing Wang ◽  
...  
Keyword(s):  
Mechanical Stress ◽  
Type Ii Collagen ◽  
Cell Types ◽  
Signal Pathways ◽  
Matrix Synthesis ◽  
Integrin Β1 ◽  
Chondrocyte Proliferation ◽  
Engineered Cartilage

Background/Aims: Periodic mechanical stress activates integrin β1-initiated signal pathways to promote chondrocyte proliferation and matrix synthesis. Integrin β1 overexpression has been demonstrated to play important roles in improving the activities and functions of several non-chondrocytic cell types. Therefore, in the current study, we evaluated the effects of integrin β1 up-regulation on periodic mechanical stress-induced chondrocyte proliferation, matrix synthesis and ERK1/2 phosphorylation in chondrocyte monolayer culture, and evaluated the quality of tissue-engineered cartilage constructed in vitro under periodic mechanical stress combined with integrin β1 up-regulation. Methods and Results: Our results revealed that under periodic mechanical stress, pre-treatment with integrin β1-wild type vector significantly enhanced chondrocyte proliferation and matrix synthesis and promoted ERK1/2 phosphorylation in comparison to mock transfectants. Furthermore, when chondrocytes were seeded in PLGA scaffolds, more accumulated GAG and type II collagen tissue were detected after Lv-integrin β1 transfection compared with sham controls exposed to periodic mechanical stress. In contrast, in the Lv-shRNA-integrin β1 group, the opposite results were observed. Conclusion: Our findings collectively suggest that in addition to periodic mechanical stress, integrin β1 up-regulation in chondrocytes could further improve the quality of tissue-engineered cartilage.

scholarly journals Periodic Mechanical Stress Activates PKCδ-Dependent EGFR Mitogenic Signals in Rat Chondrocytes via PI3K-Akt and ERK1/2

2016 ◽  
Vol 39 (4) ◽  
pp. 1281-1294 ◽  
Author(s):  
Peng He ◽  
Nan Shen ◽  
Gongming Gao ◽  
Xuefeng Jiang ◽  
Huiqing Sun ◽  
...  
Keyword(s):  
Mechanical Stress ◽  
Phosphorylation Site ◽  
Growth Factor Receptor ◽  
Cell Counting ◽  
Chondrocyte Proliferation ◽  
Extracellular Signal ◽  
Investigation Methods ◽  
Direct Cell ◽  
Sense And Respond ◽  
Static Conditions

Background/Aims: The present study aimed to analyze the mechanisms by which periodic mechanical stress is translated into biochemical signals, and to verify the important role of signaling molecules including phosphatidylinositol-3-kinase (PI3K)-Akt, protein kinase C (PKC), and epidermal growth factor receptor (EGFR) in chondrocyte proliferation. The effects of periodic mechanical stress on the mitogenesis of chondrocytes have been studied extensively in recent years. However, the mechanisms underlying the ability of chondrocytes to sense and respond to periodic mechanical stress need further investigation. Methods: Two steps were undertaken in the experiment. In the first step, the cells were pretreated with shRNA targeted to Akt or EGFR or PKCδ or control scrambled shRNA. Moreover, they were pretreated with LY294002, GF109203X, Gö6976, rottlerin, and AG1478. They were maintained under static conditions or periodic mechanical stress for 3 days, 8 h per day, prior to direct cell counting and CCK-8 assay, respectively. In the second step, the cells were pretreated with shRNA targeted to Akt or EGFR or PKCδ or control scrambled shRNA. Moreover, they were pretreated with LY294002, AG1478, and rottlerin. They were maintained under static conditions or periodic mechanical stress for 1 h prior to Western blot analysis. Results: Proliferation was inhibited by pretreatment with PKC or PKCδ inhibitor GF109203X or rottlerin and by short hairpin RNA (shRNA) targeted to PKCδ, but not by PKCα inhibitor Gö6976 in chondrocytes in response to periodic mechanical stress. Meantime, rottlerin and shRNA targeted to PKCδ also attenuated EGFR, Akt, and ERK1/2 activation. Furthermore, inhibiting EGFR activity by AG1478 and shRNA targeted to EGFR abrogated chondrocyte proliferation and phosphorylation levels of Akt and extracellular signal-regulated kinase (ERK)1/2 subjected to periodic mechanical stress, while the phosphorylation site of PKCδ was not affected. In addition, pretreatment with the PI3K-Akt-selective inhibitor LY294002 and shRNA targeted to Akt reduced periodic mechanical stress-induced chondrocyte proliferation and phosphorylation of ERK1/2, while the phosphorylation levels of EGFR and PKCδ were not inhibited. Conclusion: These findings suggested that periodic mechanical stress promoted chondrocyte proliferation through PKCδ-EGFR-PI3K-Akt-ERK1/2. They provide a stronger viewpoint for further investigations into chondrocyte mechanobiology under periodic mechanical stress and the ways to improve the quality of tissue-engineered cartilage.

scholarly journals Periodic Mechanical Stress Stimulates GIT1-Dependent Mitogenic Signals in Rat Chondrocytes Through ERK1/2 Activity

2018 ◽  
Vol 50 (3) ◽  
pp. 1015-1028 ◽  
Author(s):  
Kewei Ren ◽  
Jilei Tang ◽  
Xuefeng Jiang ◽  
Huiqing Sun ◽  
Luming Nong ◽  
...  
Keyword(s):  
Western Blot ◽  
Mechanical Stress ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Counting ◽  
Mechanical Stimuli ◽  
Wild Type ◽  
Integrin Β1 ◽  
Chondrocyte Proliferation ◽  
Blocking Antibody

Background/Aims: The mitogenic effects of periodic mechanical stress on chondrocytes have been studied extensively, but the mechanisms whereby chondrocytes sense and respond to mechanical stimuli remain to be determined. We explored the question and verified the key role of G protein coupled receptor kinase interacting protein 1 (GIT1) signaling in periodic mechanical stress-induced chondrocyte proliferation. Methods: Two steps were undertaken in the experiment. In the first step, the cells were maintained under non-pressure conditions or periodic mechanical stress for 1 h prior to Western blot analysis. In the second step, the cells were pretreated with short hairpin RNA (shRNA) targeted to GIT1 or Src or control scrambled shRNA, or transfected with GIT1 wild-type or GIT1 mutant Y321F, or focal adhesion kinase (FAK) wild-type or FAK mutants Y397F or Y576F/Y577, respectively. Moreover, the cells were pretreated with blocking antibody against integrin β1 or PP2. Then the cells were maintained under non-pressure conditions or periodic mechanical stress for 1 h prior to Western blot analysis, and for 3 days, 8 h per day, prior to direct cell counting and CCK-8 assay, respectively. Results: Periodic mechanical stress significantly induced sustained phosphorylation of GIT1 at Tyr321. Reduction of GIT1 with shRNA targeted to GIT1 and GIT1 mutant Y321F inhibited periodic mechanical stress-promoted chondrocyte proliferation, accompanied by attenuated extracellular signal-regulated kinase (ERK)1/2 and FAK phosphorylation at Tyr576/577. However, activation of Src and FAK-Tyr397 was not prevented upon GIT1 suppression. Furthermore, pretreatment with blocking antibody against integrin β1, Src-selective inhibitor, PP2, and shRNA targeted to Src blocked GIT1 activation under periodic mechanical stress. In addition, GIT1 phosphorylation at Tyr321 was not reduced upon pretreatment with FAK mutants Y397F or Y576F/Y577 under conditions of periodic mechanical stress. Conclusion: These findings collectively suggested that periodic mechanical stress promoted chondrocyte proliferation through at least two separate pathways, integrin β1-Src-GIT1-FAK(Tyr576/577)-ERK1/2, and the other parallel GIT1-independent integrin β1-FAK(Tyr397)-ERK1/2.

scholarly journals Periodic Mechanical Stress Stimulates Cav-1-Dependent IGF-1R Mitogenic Signals in Rat Chondrocytes Through ERK1/2

2018 ◽  
Vol 48 (4) ◽  
pp. 1652-1663 ◽  
Author(s):  
Kewei Ren ◽  
Jilei Tang ◽  
Xuefeng Jiang ◽  
Huiqing Sun ◽  
Luming Nong ◽  
...  
Keyword(s):  
Western Blot ◽  
Mechanical Stress ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Biological Effects ◽  
Phosphorylation Site ◽  
Cell Counting ◽  
Mechanical Stimuli ◽  
Chondrocyte Proliferation ◽  
Static Conditions

Background/Aims: The biological effects of periodic mechanical stress on the mitogenesis of chondrocytes have been studied extensively over the past few years. However, the mechanisms underlying the ability of chondrocytes to sense and respond to mechanical stimuli remain to be determined. In the current study, we analyzed the mechanisms by which periodic mechanical stress is translated into biochemical signals and verified the key role of non-integrin mechanosensors including Caveolin-1 (Cav-1), and insulin-like growth factor-1 receptor (IGF-1R) in chondrocyte proliferation. Methods: Two steps were undertaken in the experiment. In the first step, the cells were maintained under static conditions or periodic mechanical stress for 0 h and 1 h prior to Western blot analysis. In the second step, the cells were pretreated with short hairpin RNA (shRNA) targeted to Cav-1 or IGF-1R or control scrambled shRNA. Moreover, they were pretreated with their selective inhibitors methyl β-cyclodextrin (MCD) or Linsitinib (OSI-906). They were maintained under static conditions or periodic mechanical stress for 1 h prior to Western blot analysis, and for 3 days, 8 h per day, prior to direct cell counting and CCK-8 assay, respectively. Results: Periodic mechanical stress significantly induced sustained phosphorylation of Cav-1 at Tyr14 and IGF-1R at Tyr1135/1136. Proliferation was inhibited by pretreatment with Cav-1 inhibitor MCD and by shRNA targeted to Cav-1 in chondrocytes in response to periodic mechanical stress. Meantime, MCD and shRNA targeted to Cav-1 also attenuated IGF-1R, and extracellular signal-regulated kinase (ERK)1/2 activation. In addition, inhibiting IGF-1R activity by Linsitinib and shRNA targeted to IGF-1R abrogated chondrocyte proliferation and phosphorylation level of ERK1/2 subjected to periodic mechanical stress, while the phosphorylation site of Cav-1 was not affected. Conclusion: These findings collectively suggested that periodic mechanical stress promoted chondrocyte proliferation through Cav-1-IGF-1R-ERK1/2.

scholarly journals Appropriate Reference Genes for RT-qPCR Normalization in Various Organs of Anemone Flaccida Fr. Schmidt at Different Growing Stages

Genes ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 459
Author(s):  
Zeying Zhao ◽  
Hanwen Zhou ◽  
Zhongnan Nie ◽  
Xuekui Wang ◽  
Biaobiao Luo ◽  
...  
Keyword(s):  
Reference Genes ◽  
Gene Selection ◽  
Quantitative Polymerase Chain Reaction ◽  
Cultivated Plants ◽  
Gene Expressions ◽  
Natural Habitats ◽  
Triterpenoid Saponins ◽  
Qpcr Normalization ◽  
Suitable Reference

Anemone flaccida Fr. Schmidt is a traditional medicinal herb in southwestern China and has multiple pharmacological effects on bruise injuries and rheumatoid arthritis (RA). A new drug with a good curative effect on RA has recently been developed from the extract of A. flaccida rhizomes, of which the main medicinal ingredients are triterpenoid saponins. Due to excessive exploitation, the wild population has been scarce and endangered in a few of its natural habitats and research on the cultivation of the plant commenced. Studies on the gene expressions related to the biosynthesis of triterpenoid saponins are not only helpful for understanding the effects of environmental factors on the medicinal ingredient accumulations but also necessary for monitoring the herb quality of the cultivated plants. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) as a sensitive and powerful technique has been widely used to detect gene expression across tissues in plants at different stages; however, its accuracy and reliability depend largely on the reference gene selection. In this study, the expressions of 10 candidate reference genes were evaluated in various organs of the wild and cultivated plants at different stages, using the algorithms of geNorm, NormFinder and BestKeeper, respectively. The purpose of this study was to identify the suitable reference genes for RT-qPCR detection in A. flaccida. The results showed that two reference genes were sufficient for RT-qPCR data normalization in A. flaccida. PUBQ and ETIF1a can be used as suitable reference genes in most organs at various stages because of their expression stabilitywhereas the PUBQ and EF1Α genes were desirable in the rhizomes of the plant at the vegetative stage.

BMP2 Is Required for Postnatal Maintenance of Osteochondral Tissues of the Temporomandibular Joint

Cartilage ◽  
2020 ◽  
pp. 194760352098015
Author(s):  
Mara H. O’Brien ◽  
Eliane H. Dutra ◽  
Shivam Mehta ◽  
Po-Jung Chen ◽  
Sumit Yadav
Keyword(s):  
Age Groups ◽  
Mandibular Condyle ◽  
Bone Morphogenetic Protein 2 ◽  
Matrix Synthesis ◽  
Cartilage Growth ◽  
Chondrocyte Proliferation ◽  
Conditional Deletion ◽  
Morphogenetic Protein

Objective Bone morphogenetic protein 2 (BMP2) plays important roles in cartilage growth and development. Paradoxically, elevated levels of BMP2 leads to hypertrophic differentiation and osteoarthritis of cartilage. We examined the in vivo loss of BMP2 in cells expressing aggrecan of the mandibular condyle and knee. Design Three-week-old BMP2 flox/flox- CreER-positive mice and their Cre-negative littermates were treated with tamoxifen and raised until 3 or 6 months. We also investigated the direct effects of BMP2 on chondrocytes in vitro. Cells from the mandibular condyle of mice were treated with recombinant human BMP2 (rhBMP2) or rhNoggin (inhibitor of BMP2 signaling). Results Conditional deletion of BMP2 caused breakage of the cartilage integrity in the mandibular condyle of mice from both age groups, accompanied by a decrease in cartilage thickness, matrix synthesis, mineralization, chondrocyte proliferation, and increased expression of degeneration markers, while the effects at articular cartilage were not significant. In vitro results revealed that rhBMP2 increased chondrocyte proliferation, mineralization, and differentiation, while noggin induced opposite effects. Conclusions In conclusion, BMP2 is essential for postnatal maintenance of the osteochondral tissues of the mandibular condyle.

scholarly journals Transient Supplementation of Anabolic Growth Factors Rapidly Stimulates Matrix Synthesis in Engineered Cartilage

2011 ◽  
Vol 39 (10) ◽  
pp. 2491-2500 ◽  
Author(s):  
Kenneth W. Ng ◽  
Christopher J. O’Conor ◽  
Lindsay E. Kugler ◽  
James L. Cook ◽  
Gerard A. Ateshian ◽  
...  
Keyword(s):  
Growth Factors ◽  
Matrix Synthesis ◽  
Engineered Cartilage

Mechanical quality of tissue engineered cartilage: Results after 6 and 12 weeksin vivo

2000 ◽  
Vol 53 (6) ◽  
pp. 673-677 ◽  
Author(s):  
Georg N. Duda ◽  
Andreas Haisch ◽  
Michaela Endres ◽  
Christian Gebert ◽  
Daniel Schroeder ◽  
...  
Keyword(s):  
Mechanical Quality ◽  
Engineered Cartilage

Methodological Implications on Quantitative Studies of Cytocompatibility in Direct Contact with Bioceramic Surfaces

2011 ◽  
Vol 493-494 ◽  
pp. 325-330 ◽  
Author(s):  
J.A. Cortês ◽  
Elena Mavropoulos ◽  
Moema Hausen ◽  
Alexandre Rossi ◽  
J.M. Granjeiro ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Crystal Violet ◽  
Fluorescent Dyes ◽  
Ceramic Materials ◽  
Neutral Red ◽  
Cell Counting ◽  
Cell Detection ◽  
Porous Hydroxyapatite ◽  
Detection And Counting ◽  
Direct Cell

Cell adhesion, proliferation and differentiation are important specific parameters to be evaluated on biocompatibility studies of candidate biomaterials for clinical applications. Several different methodologies have been employed to study, both qualitative and quantitatively, the direct interactions of ceramic materials with cultured mammal and human cells. However, while quantitatively evaluating cell density, viability and metabolic responses to test materials, several methodological challenges may arise, either by impairing the use of some widely applied techniques, or by generating false or conflicting results. In this work, we tested the inherent interference of different representative calcium phosphate ceramic surfaces (stoichiometric dense and porous hydroxyapatite (HA) and cation-substituted apatite tablets) on different tests for quantitative evaluation of osteoblast adhesion and metabolism, either based on direct cell counting after trypsinization, colorimetric assays (XTT, Neutral Red and Crystal Violet) and fluorescence microscopy. Cell adhesion estimation after trypsinization was highly dependent on the time of treatment, and the group with the highest level of estimated adhesion was inverted from 5 to 20 minutes of exposition to trypsin. Both dense and porous HA samples presented high levels of background adsorption of the Crystal Violet dye, impairing cell detection. HA surfaces also were able to adsorb high levels of fluorescent dyes (DAPI and phalloidin-TRITC), generating backgrounds which, in the case of porous HA, impaired cell detection and counting by image processing software (Image Pro Plus 6.0). We conclude that the choice for the most suitable method for cell detection and estimation is highly dependent on very specific characteristics of the studied material, and methodological adaptations on well established protocols must always be carefully taken on consideration.

Single and Few-Layer MoS2: CVD Synthesis, Transference, and Photodetection Application

MRS Advances ◽  
2017 ◽  
Vol 2 (60) ◽  
pp. 3709-3714
Author(s):  
Gustavo A. Saenz ◽  
Carlos de Anda Orea ◽  
Anupama B. Kaul
Keyword(s):  
Chemical Vapor Deposition ◽  
Vapor Deposition ◽  
Flexible Substrate ◽  
Synthesis Method ◽  
Chemical Vapor ◽  
Synthesis Methods ◽  
Phase Reaction ◽  
Isolation And Characterization ◽  
Cvd Synthesis

ABSTRACTTwo-dimensional layered materials, materials with weak out-of-plane van der Waals bonding and strong in-plane covalent bonding, have attracted special attention in recent years since the isolation and characterization of monolayer graphite, the graphene. The electrical bandgap in Transition Metal Di-Chalcogenides (TMDCs), non-existent in graphene, make them a good alternative family of materials for novel electronic and optoelectronic applications. 2H- MoS2, one of the most stable TMDCs, has been extensively studied, including the synthesis methods, and its potential applications in photodetection. The chemical vapor deposition (CVD) synthesis method has increased its potential over the years. The advantages of this method are scalability compared to micromechanical exfoliation, common process used in research laboratories, and the maintenance of the quality and intrinsic properties of the material compared to the liquid exfoliation methods. In this work, we synthesized high quality pristine 2H-MoS2 via atmospheric pressure chemical vapor deposition (APCVD) by vapor phase reaction of MoO3 and S powder precursors. The samples were characterized via Raman and photoluminescence (PL) spectroscopy and compared to mechanically exfoliated MoS2 crystal by measuring the full-width half maxima (FWHM) of monolayer and few-layer mesoscopic flakes. In addition, the CVD synthesized single and few-layered MoS2 domains were transferred to different substrates using a high yield process, including a flexible substrate, preserving the quality of the material. Finally, and mechanically exfoliated MoS2 two-terminal photodetector was designed, fabricated, and measured. Demonstrating thus the capability of heterostructure fabrication and the quality of our synthesis and device fabrication process.

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