Intracellular calcium ([Ca2+]i) is a second messenger molecule critical for numerous intracellular signaling pathways in endothelial cells (ECs). Direct mechanical stimulation imposed on single ECs by a microprobe has been demonstrated to increase [Ca2+]i levels in ECs. After an initial delay time, this Ca2+-signal propagates from the mechanically stimulated cell to its neighboring cells in the form of an intercellular Ca2+-wave, a process termed intercellular communication. Although intercellular communication is a fundamental property of many multicellular systems, it remains unclear as to whether intercellular communication following shear stress of ECs actually occurs, and if so, whether this communication occurs via gap junctions or the release of extracellular mediators including ATP. In the current study, we investigated ATP release from ECs during periods of shear stress and measured intercellular Ca2+-waves using adenosine 5'-triphosphate, P3-(1-(2-nitrophenyl)ethyl)ester and disodium salt (NPE-caged ATP) stimulation. In addition, we investigated intercellular communication in ECs during shear stress using chemical inhibitors of both gap junctions and various components of the ATP paracrine signaling pathway. ECs subjected to shear stress loading released ATP. Using NPE-caged ATP, local increase of extracellular ATP induced a Ca2+-wave. Furthermore, [Ca2+]i responses in ECs under shear stress loading was inhibited by the purinergic receptor blocker, ATPase, and several metabolic inhibitors including FCCP and rotenone. These results suggest that [Ca2+]i communication mediated by ATP exists in ECs under shear stress loading in vitro.

Keywords

Endothelial Cell, Shear Stress, ATP, Autocrine/Paracrine, Calcium Signaling

Paper information

Susumu KUDO, Kaoru HOSOE, Makoto HOSOBUCHI, Naoto KAWASAKI and Kazuo TANISHITA, “ATP Release from Cultured Endothelial Cells and Intercellular Calcium Signaling during Shear Stress Exposure”, Journal of Biomechanical Science and Engineering, Vol. 4, No. 2 (2009), pp.274-285 . doi:10.1299/jbse.4.274

The effects of three-dimensional (3-D) culture and cyclic stretch stimulation on the expression of contractile proteins were investigated in freshly isolated rat aortic smooth muscle cells (FSMC). Primary cells were cultured statically on cell culture dishes (two-dimensional (2-D) culture) or in type I collagen gel matrix (3-D culture). Changes in their expression level of actin filaments (AFs) and smooth muscle myosin heavy chain (SM-MHC) were measured quantitatively using an accurately-calibrated fluorescent microscopy. The expression of AFs and SM-MHC decreased in both cultures in their early stages. Cell morphology was quite different between the two cultures: the cells had a flattened and irregular shape in the 2-D culture, while they had a fusiform shape with a well-defined long axis in the 3-D. Nineteen-day culture in the gel significantly increased the expression levels of AFs and SM-MHC while the expression levels remained low in the 2-D. Further and early increase in the expression levels was observed in the cells cultured in the gel with cyclic stretch of ?8% amplitude and 1 Hz frequency. The cyclic stretch also induced alignment of FSMCs in the gel parallel to the stretch direction, and the cell alignment was observed earlier than the increase in their contractile proteins. These results indicate that the 3-D culture in collagen gel may increase the expression level of contractile proteins in FSMCs while maintaining their fusiform morphology, and cyclic stretch may efficiently increase the expression levels when the cells aligned in the stretch direction.

Keywords

Cellular Biomechanics, Smooth Muscle Phenotype, Contractile Properties, Extracellular Matrix, Actin Stress Fibers

Paper information

Kazuaki NAGAYAMA, Naoki MORISHIMA and Takeo MATSUMOTO, “Effects of Three-Dimensional Culture and Cyclic Stretch Stimulation on Expression of Contractile Proteins in Freshly Isolated Rat Aortic Smooth Muscle Cells”, Journal of Biomechanical Science and Engineering, Vol. 4, No. 2 (2009), pp.286-297 . doi:10.1299/jbse.4.286

The objective of this study is to analyze kinetics and kinematics of artificial knee joint at deep flexion, using a 2D geometric model. Our knee model was composed of the tibio-femoral and patello-femoral joints, including the patella tendon and the quadriceps muscles. Since the model was 2D, it neglected the rotation and the varus/valgus motion. In the model, such assumption was made that articulation surfaces were rigid and a point contact was made. Also it was assumed that the force and moment equilibrium conditions were always satisfied. The motion to be studied was decided as the motion from standing to deep squatting with heel-rising. From the simulation we found such results that the femur did not make a rollback and contact force at post-cam became pretty large in high and deep flexion, indicating the post-cam mechanism did not function well. Also patello-femoral contact forces reached larger than tibio-femoral contact forces. Our results demonstrated that how to design the post-cam and patello-femoral joint should be critical issues for developing a new prosthesis which can make deep flexion.

Keywords

Artificial Knee Joint, Deep Knee Flexion, 2D Model Analysis

Paper information

Michihiko FUKUNAGA, Tadasuke KATSUHARA, Shunji HIROKAWA, Masaaki MAWATARI and Takao HOTOKEBUCHI, “A 2D Model Analysis of Artificial Knee Joint during Deep Squatting”, Journal of Biomechanical Science and Engineering, Vol. 4, No. 2 (2009), pp.298-305 . doi:10.1299/jbse.4.298