Application of hydrostatic pressure up-regulates sost gene expression in osteocytic spheroids.

In this study, we developed a hydrostatic pressurizing chamber capable of applying hydrostatic pressure to osteocytic spheroids derived from mouse osteoblastic MC3T3-E1 cells. Our results demonstrate that a 4-hour exposure to 200 kPa of hydrostatic pressure did not alter the apparent morphology of the spheroids. However, gene expression analysis revealed a significant up-regulation of Sost, marker of late-stage osteocyte differentiation.

Macroscopic creep behavior of spheroids derived from mesenchymal stem cells under compression.

Spheroid culture, where cells are aggregated three-dimensionally, is expected to have applications as a model that better recapitulates invivo environment beyond two-dimensional environments. When human mesenchymal stem cells are subjected to spheroid culture in the presence of osteogenesis supplements, the gene expression of osteocyte differentiation marker is greatly increased within a short period compared to two-dimensional culture. However, how such alterations may be reflected to mechanical properties of the spheroid remains unknown.

Phosphate overload via the type III Na-dependent Pi transporter represses aortic wall elastic fiber formation.

Objectives: Phosphate (Pi) induces differentiation of arterial smooth muscle cells to the osteoblastic phenotype by inducing the type III Na-dependent Pi transporter Pit-1/solute carrier family member 1. This induction can contribute to arterial calcification, but precisely how Pi stress acts on the vascular wall remains unclear. We investigated the role of extracellular Pi in inducing microstructural changes in the arterial wall.

Spatiotemporal analysis of multi-scale cell structure in spheroid culture reveals hypertrophic chondrocyte differentiation.

3D cell culture has emerged as a promising approach to replicate the complex behaviors of cells within living organisms. This study aims to analyze spatiotemporal behavior of the morphological characteristics of cell structure at multiscale in 3D scaffold-free spheroids using chondrogenic progenitor ATDC5 cells. Over a 14-day culture period, it exhibited cell hypertrophy in the spheroids regarding cellular and nuclear size as well as changes in morphology.

Novel air-liquid interface culture model to investigate stiffness-dependent behaviors of alveolar epithelial cells.

Pulmonary alveoli are functional units in gas exchange in the lung, and their dysfunctions in lung diseases such as interstitial pneumonia are accompanied by fibrotic changes in structure, elevating the stiffness of extracellular matrix components. The present study aimed to test the hypothesis that such changes in alveoli stiffness induce functional alteration of epithelial cell functions, exacerbating lung diseases. For this, we have developed a novel method of culturing alveolar epithelial cells on polyacrylamide gel with different elastic modulus at an air-liquid interface.

Contributions of collagen and elastin to elastic behaviours of tendon fascicle.

Tendon exhibits the capacity to be stretched and to return to its original length without suffering structural damage in vivo, a capacity known as elastic recoil. Collagen fibres are aligned longitudinally and elastin fibres mostly run parallel to collagen fibres in tendon. However, their interactions and contributions to tendon elastic behaviours are not well understood.

In situ FRET measurement of cellular tension using conventional confocal laser microscopy in newly established reporter mice expressing actinin tension sensor.

FRET-based sensors are utilized for real-time measurements of cellular tension. However, transfection of the sensor gene shows low efficacy and is only effective for a short period. Reporter mice expressing such sensors have been developed, but sensor fluorescence has not been measured successfully using conventional confocal microscopy.

A posterior tibial slope angle over 12 degrees is critical to epiphyseal fracture of the proximal tibia: Three-dimensional finite element analysis.

Introduction: The effects of the proximal tibial slope angle on the proximal tibial epiphysis remain unknown. To elucidate those effects, we investigated the strain distribution in proximal tibial epiphysis with different proximal tibial slope angles and proximal tibial epiphysis closure periods using finite element analysis.

Materials And Methods: The finite element models of the proximal tibia were reconstructed from CT images and consisted of cancellous/cortical bone and epiphyseal plate.

3D quantitative assessment for nuclear morphology in osteocytic spheroid with optical clearing technique.

In recent years, three-dimensional (3D) cell culture has been attracting attention as a cell culture model that mimics an environment closer to that of a living organism. It is known that there is a close relationship between cell nuclear shape and cellular function, which highlights the importance of cell nucleus shape analysis in the 3D culture. On the other hand, it is difficult to observe the cell nuclei inside the 3D culture models because the penetration depth of the laser light under a microscope is limited.

Androgen promotes squamous differentiation of atypical cells in cervical intraepithelial neoplasia via an ELF3-dependent pathway.

Background: Since the human papillomavirus vaccines do not eliminate preexisting infections, nonsurgical alternative approaches to cervical intraepithelial neoplasia (CIN) have been required. We previously reported that FOXP4 (forkhead box transcription factor P4) promoted proliferation and inhibited squamous differentiation of CIN1-derived W12 cells. Since it was reported that FOXP expressions were regulated by the androgen/androgen receptor (AR) complex and AR was expressed on the CIN lesions, in this study we examined the effects of androgen on CIN progression.

Changes in the intra- and extra-mechanical environment of the nucleus in Saos-2 osteoblastic cells during bone differentiation process: Nuclear shrinkage and stiffening in cell differentiation.

Osteogenic differentiation has been reportedly regulated by various mechanical stresses, including fluid shear stress and tensile and compressive loading. The promotion of osteoblastic differentiation by these mechanical stresses is accompanied by reorganization of the F-actin cytoskeleton, which is deeply involved in intracellular forces and the mechanical environment. However, there is limited information about the effect on the mechanical environment of the intracellular nucleus, such as the mechanical properties of the nucleus and intracellular forces exerted on the nucleus, which have recently been found to be directly involved in various cellular functions.

Spheroid culture for chondrocytes triggers the initial stage of endochondral ossification.

Endochondral ossification is the process of bone formation derived from growing cartilage duringskeletal development. In previous studies, we provoked the osteocyte differentiation of osteoblast precursor cells under a three-dimensional (3D) culture model. To recapitulate the endochondral ossification, the present study utilized the self-organized scaffold-free spheroid model reconstructed by pre-chondrocyte cells.

Effect of chemically induced osteogenesis supplements on multicellular behavior of osteocytic spheroids.

Understanding in multicellular behaviors in three-dimensional (3D) culture models such as organoids is important to help us better comprehend the mechanisms of the morphogenesis and functions of diverse organs in vivo cellular environment. In this study, we elucidated the multicellular behaviors of the osteocytic spheroids in response to the chemically induced osteogenesis supplements (OS). Particularly, we conducted 1) size change measurement, 2) fusion experiment, and 3) collagen embedding experiment of spheroids, in response to the OS.

FOXP4 inhibits squamous differentiation of atypical cells in cervical intraepithelial neoplasia via an ELF3-dependent pathway.

Although the human papillomavirus (HPV) vaccine is effective for preventing cervical cancers, this vaccine does not eliminate pre-existing infections, and alternative strategies have been warranted. Here, we report that FOXP4 is a new target molecule for differentiation therapy of cervical intraepithelial neoplasia (CIN). An immunohistochemical study showed that FOXP4 was expressed in columnar epithelial, reserve, and immature squamous cells, but not in mature squamous cells of the normal uterine cervix.

Stiffness estimation of transversely anisotropic materials using a novel indentation tester with a rectangular hole.

Since embryos change their morphology drastically in the gastrulation stage, mechanical characterization of young embryos is important as they also change their tissue stiffness with the stage of development. Herein, virtual compression tests were conducted assuming that the gastrula has a spherical shape with transverse anisotropy. Based on the design of experiments, we found that the Young's moduli and material anisotropy can be efficiently determined by measuring the reaction force and surface displacement when indenting the tester into an embryo.

Dependency of deformation of cell nucleus on stretch direction of tissue: Relation to anisotropic response of aortic media to hypertension.

The deformation of the cell nucleus may cause dispersion of chromatin and eventually enhance transcription, translation, and protein expression. If this happens in the hypertensive artery, an excessive stretch of smooth muscle cell (SMC) nuclei caused by hypertension may provoke wall thickening. Here, we measured deformation of SMC nuclei in rabbit thoracic aortas stretched in different directions.

Rapid fabrication of tendon-like collagen gel via simultaneous fibre alignment and intermolecular cross-linking under mechanical loading.

Artificial tissue replacement is a promising strategy for better healing outcomes for tendon and ligament injuries, due to the very limited self-regeneration capacity of these tissues in mammals, including humans. Because clinically available synthetic and biological scaffolds for tendon repair have performed more poorly than autografts, both biological and mechanical compatibility need to be improved. Here we propose a rapid fabrication method for tendon-like structure from collagen hydrogel, simultaneously achieving collagen fibre alignment and intermolecular cross-linking.

Through the cleared aorta: three-dimensional characterization of mechanical behaviors of rat thoracic aorta under intraluminal pressurization using optical clearing method.

The media of aortic wall is characterized by altering layers of elastin and smooth muscle cells (SMCs), along with collagen fibers in both layers, and plays a central role in functional and pathological remodeling such as hypertension and atherosclerosis. Because the arterial function is linked closely to the arterial wall internal structure, it is essential to investigate the alteration of the arterial microstructure during macroscopic deformation to understand cardiovascular pathologies. The present study adopted a tissue clearing method in three-dimensional mechanical characterization of rat thoracic aorta, and successfully observed changes in the structure of each of the three primary components of the aorta under intraluminal pressurization while maintaining tissue mechanical integrity and flexibility.

Microscopic characterisation of local strain field in healing tissue in the central third defect of mouse patellar tendon at early-phase of healing.

Tendons exhibit a hierarchical collagen structure, wherein higher-level components, such as collagen fibres and fascicles, are elongated, slid, and rotated during macroscopic stretching. These mechanical behaviours of collagen fibres play important roles in stimulating tenocytes, imposing stretching, compression, and shear deformation. It was hypothesised that a lack of local fibre behaviours in healing tendon tissue may result in a limited application of mechanical stimuli to cells within the tissue, leading to incomplete recovery of tissue structure and functions in regenerated tendons.

A novel third mesh-like myometrial layer connects the longitudinal and circular muscle fibers -A potential stratum to coordinate uterine contractions.

Periodic myometrial contraction is one of the important uterine functions to achieve embryo implantation and parturition. Although it is well-known that the mammalian myometrium is composed of longitudinal (outer) and circular (inner) layers, the precise mechanisms that coordinate both muscular contractions to produce peristaltic movements remain unclear. Recently, by treatment with our modified Clear Unobstructed Brain Imaging Cocktails and Computational analysis (CUBIC) tissue-clearing method, we obtained well-contrasted three-dimensional images of the transparent murine ovary using enhanced green fluorescent protein (EGFP) transgenic mice and light-sheet microscopy.

Three-Dimensional Finite Analysis of the Optimal Alignment of the Tibial Implant in Unicompartmental Knee Arthroplasty.

Background: Although unicompartmental knee arthroplasty (UKA) has become more common because of its good outcomes, several complications have been reported. Tibial implant alignment, an important cause of such complications, has been investigated; however, the optimal alignment of the tibial implant has not been determined. This study used 3-dimensional finite element analysis to investigate changes in stress distribution in the proximal tibia after UKA at multiple tibial implant alignments.