Real-time imaging of intracellular deformation dynamics in vibrated adherent cell cultures.

Mechanical vibration has been shown to regulate cell proliferation and differentiation in vitro and in vivo. However, the mechanism of its cellular mechanotransduction remains unclear. Although the measurement of intracellular deformation dynamics under mechanical vibration could reveal more detailed mechanisms, corroborating experimental evidence is lacking due to technical difficulties. In this study, we aimed to propose a real-time imaging method of intracellular structure deformation dynamics in vibrated adherent cell cultures and investigate whether organelles such as actin filaments connected to a nucleus and the nucleus itself show deformation under horizontal mechanical vibration. The proposed real-time imaging was achieved by conducting vibration isolation and making design improvements to the experimental setup; using a high-speed and high-sensitivity camera with a global shutter; and reducing image blur using a stroboscope technique. Using our system, we successfully produced the first experimental report on the existence of the deformation of organelles connected to a nucleus and the nucleus itself under horizontal mechanical vibration. Furthermore, the intracellular deformation difference between HeLa and MC3T3-E1 cells measured under horizontal mechanical vibration agrees with the prediction of their intracellular structure based on the mechanical vibration theory. These results provide new findings about the cellular mechanotransduction mechanism under mechanical vibration.

A concept for emotion recognition systems for children with profound intellectual and multiple disabilities based on artificial intelligence using physiological and motion signals.

PURPOSE: This study proposes a concept for emotion recognition systems for children with profound intellectual and multiple disabilities (PIMD) based on artificial intelligence (AI) using physiological and motion signals. METHODS: First, the heartbeat interval (R-R interval, RRI) of a child with PIMD was measured, and the correlation between the RRI and emotion was briefly tested in a preliminary experiment. Then, a concept based on AI for emotion recognition systems for children with PIMD was created using physiological and motion signals, and an emotion recognition system based on the proposed concept was developed using a random forest classifier taking as inputs the RRI, eye gaze, and other data acquired using low physical burden sensors. Subsequently, the developed emotion recognition system was evaluated, validating the proposed concept. Finally, we proposed a validated concept for emotion recognition systems. RESULTS: A correlation was found between the RRI and emotion. The emotion recognition system was created based on the proposed concept and tested. According to the results, the recognition rate of "negative" and "not negative" of 70.4% ± 6.1% (Mean ± S.D.) of the developed emotion recognition system was higher than 48.5% ± 5.0% of an unfamiliar person used as a control. CONCLUSION: The results indicate that the proposed concept for emotion recognition systems is useful for communicating with children with PIMD.

A new concept based on artificial intelligence for emotion recognition systems for children with profound intellectual and multiple disabilities (PIMD) using physiological and motion signals is proposed.An emotion recognition system based on the proposed concept developed using a random forest classifier taking as inputs the heartbeat interval, eye gaze, and other data acquired using low physical burden sensors were tested in terms of the emotion recognition rate.The recognition rate of "negative" and "not negative" of the developed system (i.e., 70.4% ± 6.1%) is higher than that of an unfamiliar person (i.e., 48.5% ± 5.0%).The proposed concept for emotion recognition systems may be useful for communicating with children with PIMD.

In vitro stability of open wedge high tibial osteotomy with synthetic bone graft.

It has been predicted that significant stress will be applied to the plate and lateral cortical hinge of an osteotomy site when early full weight bearing is commenced after an open wedge high tibial osteotomy. We hypothesized that the stress concentration on the plate or at the lateral cortical hinge would be reduced by inserting bone substitutes into the osteotomy gap. Two different types of tibia model were investigated: Group A, fixation with TomoFix with the osteotomy site left as an open space; and Group B, two beta-TCP wedges are inserted into osteotomy site and fixed with TomoFix. Stress at five points was measured using strain gauges. Specimens were mounted onto a testing machine with an FTA (femoro-tibial angle) of 170 degrees . Cyclic load tests and an ultimate load test were then performed. The mean stress on the plate was measured at 15.5+/-1.8Mpa in Group A. On the other hand, this value in Group B was only 9.52+/-2.1Mpa and this was a significant difference (P<0.01). The mean stress on the lateral hinge in Groups A and B was 3.31+/-0.5 and 2.49+/-0.2, respectively which was also a significant difference (P<0.05). The mean maximum breaking load in Group A was 2500+/-280N and in Group B 4270+/-420N which was a significant difference (P<0.01). Hence, for OWHTO procedures, the use of beta-TCP wedges and TomoFix is thus likely to improve the initial axial and possibly rotational stability at the osteotomy site in comparison with methods that leave the osteotomy gap open.

Low-intensity pulsed ultrasound activates the phosphatidylinositol 3 kinase/Akt pathway and stimulates the growth of chondrocytes in three-dimensional cultures: a basic science study.

INTRODUCTION: The effect of low-intensity pulsed ultrasound (LIPUS) on cell growth was examined in three-dimensional-cultured chondrocytes with a collagen sponge. To elucidate the mechanisms underlying the mechanical activation of chondrocytes, intracellular signaling pathways through the Ras/mitogen-activated protein kinase (MAPK) and the integrin/phosphatidylinositol 3 kinase (PI3K)/Akt pathways as well as proteins involved in proliferation of chondrocytes were examined in LIPUS-treated chondrocytes. METHODS: Articular cartilage tissue was obtained from the metatarso-phalangeal joints of freshly sacrificed pigs. Isolated chondrocytes mixed with collagen gel and culture medium composites were added to type-I collagen honeycomb sponges. Experimental cells were cultured with daily 20-minute exposures to LIPUS. The chondrocytes proliferated and a collagenous matrix was formed on the surface of the sponge. Cell counting, histological examinations, immunohistochemical analyses and western blotting analysis were performed. RESULTS: The rate of chondrocyte proliferation was slightly but significantly higher in the LIPUS group in comparison with the control group during the 2-week culture period. Western blot analysis showed intense staining of type-IX collagen, cyclin B1 and cyclin D1, phosphorylated focal adhesion kinase, and phosphorylated Akt in the LIPUS group in comparison with the control group. No differences were detected, however, in the MAPK, phosphorylated MAPK and type-II collagen levels. CONCLUSION: LIPUS promoted the proliferation of cultured chondrocytes and the production of type-IX collagen in a three-dimensional culture using a collagen sponge. In addition, the anabolic LIPUS signal transduction to the nucleus via the integrin/phosphatidylinositol 3-OH kinase/Akt pathway rather than the integrin/MAPK pathway was generally associated with cell proliferation.

Effects of vibration and hyaluronic acid on activation of three-dimensional cultured chondrocytes.

OBJECTIVE: To investigate the effects of vibration (Vib) and hyaluronic acid (HA) on 3-dimensional cultured cartilage. METHODS: Chondrocytes were obtained from metatarsophalangeal joints of freshly killed 6-month-old pigs. Twenty-four-well plates containing type I collagen sponge disks were used to culture samples. The frequency and the amplitude of the vibration of the well plate were 100 Hz and 0.5 nm, respectively. We produced 3-dimensional cartilage tissue using HA and vibration with collagen sponge as a carrier. Four different culture conditions were examined: a control HA-Vib- group, an HA-Vib+ group, an HA+Vib- group, and an HA+Vib+ group. Each group was cultured for 2 weeks. After culture days 3, 7, 10, and 14 (every 3.5 days), the levels of chondroitin 4-sulfate (C4S) and chondroitin 6-sulfate (C6S) isomers synthesized in each culture medium were measured. Histologic analysis, immunohistochemical analysis, and electron microscopic examination were performed. RESULTS: Mean C4S and C6S synthesis had increased rapidly after 7 days of culture and continued to increase thereafter. There were significant differences among the 4 groups (P < 0.01). Synthesis of both C4S and C6S was most abundant in the HA+Vib+ group and the lowest in the HA-Vib- group. After 1 and 2 weeks of culture, the chondrocytes had formed stratified structures on the collagen sponges in all groups, although the thickest structure was observed in the HA+Vib+ group and the thinnest in the HA-Vib- group. Under immunofluorescence, the HA+Vib+ group exhibited the strongest chromatic features. Under electron microscopy, the chondrocytes in the HA+Vib+ group exhibited many long and slender prominences on their surface, and extracellular substance could be observed associated with the cells. CONCLUSION: Our results indicate that the combination of vibration and HA activates the production of proteoglycan in 3-dimensional cultured chondrocytes and stimulates MAPK and beta-catenin. This suggests that some mechanoreceptors for vibration exist on the plasma membrane of chondrocytes and activate the intracellular signal transduction system.

Identification of two differentially regulated isoforms of protochlorophyllide oxidoreductase (POR) from tobacco revealed a wide variety of light- and development-dependent regulations of POR gene expression among angiosperms.

NADPH-protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent reduction of protochlorophyllide a in the chlorophyll biosynthetic pathway. Here, we identified two distinct POR cDNAs from tobacco. Both POR isoforms are encoded by a respective single copy gene in tobacco genome. The overall deduced amino acid sequences of two tobacco cDNAs, designated here POR1 and POR2, displayed significant identities ( approximately 75%), but showed different patterns of light and developmental regulation. In contrast to the previously isolated POR isoforms of Arabidopsis thaliana and barley, the expression of both tobacco POR isoforms were not negatively regulated by light and persisted in matured green tissues. Furthermore, the expression of both genes appeared to be regulated by a diurnal regulation. These results show a wide variety of light- and development-dependent regulations of POR gene expression among angiosperms. Furthermore, phylogenetic analysis including tobacco revealed that POR gene family is differentially represented by angiosperms, most of which is probably caused by independent gene duplication in individual plant. Present results imply a modification of the previous concept that chlorophyll biosynthesis and chloroplast differentiation in angiosperms are ubiquitously controlled by unique functions of two POR isoforms.