Modeling trabecular bone adaptation to local bending load regulated by mechanosensing osteocytes

Cancellous bone has a complicated three-dimensional porous microstructure that consists of strut-like or plate-like trabeculae. The arrangement of the trabeculae is remodeled throughout the organism’s lifetime to functionally adapt to the surrounding mechanical environment. During bone remodeling, osteocytes buried in the bone matrix are believed to play a pivotal role as mechanosensory cells and help regulate the coupling of osteoclastic bone resorption and osteoblastic bone formation according to the mechanical stimuli. Previously, we constructed a mathematical model of trabecular bone remodeling incorporating cellular mechanosensing and intercellular signal transmission, in which osteocytes are assumed to sense the flow of interstitial fluid as a mechanical stimulus that regulates bone remodeling. Our remodeling simulation could describe the reorientation of a single strut-like trabecula under uniaxial loading. In the present study, to investigate the effects of a bending load on trabecular bone remodeling, we simulated the morphological change in a single trabecula under a cyclic bending load based on our mathematical model. The simulation results showed that the application of the bending load influences not only the formation of the plate-like trabecula but also the changes in trabecular topology. These results suggest the possibility that the characteristic trabecular morphology, such as the strut-like or plate-like form, is determined depending on the local mechanical environment

Investigation of mechanosensing and mechanoresponse mechanisms in osteoblasts and osteocytes: in vitro experiments targeting subcellular components

To understand adaptive bone remodeling in response to external mechanical stimuli, researchers have elucidated the mechanisms of mechanosensing and mechanoresponse through in vitro experiments targeting subcellular components from molecules to organelles. Such subcellular experiments have been performed by applying mechanical stimuli to mechanosensitive components and by measuring and observing the dynamic behaviors of the mechanosensitive and mechanoresponsive components. For a better understanding of the importance of the subcellular experiments, this article reviews the recent subcellular experiments for osteoblasts and osteocytes. First, we introduce the tools used for the stimulation and measurement/observation, and we discuss how these tools have contributed to the elucidation of the mechanisms. Second, we shed light on how the findings on the behaviors of the subcellular components have enhanced our basic understanding of the underlying mechanisms. Furthermore, we present future perspectives for subcellular experiments. To do this, we discuss the utilization of microscopes with higher spatial resolution and discuss focus points for a clearer understanding of these mechanisms in osteocytes. Future experiments will reveal how osteoblasts and osteocytes sense and respond to external mechanical stimuli in their surrounding environment in bone, and how cellular behaviors finally lead to the regulation of bone resorption and formation in adaptive bone remodeling

Interstitial fluid flow in canaliculi as a mechanical stimulus for cancellous bone remodeling: in silico validation

Cancellous bone has a dynamic 3-dimensional architecture of trabeculae, the arrangement of which is continually reorganized via bone remodeling to adapt to the mechanical environment. Osteocytes are currently believed to be the major mechanosensory cells and to regulate osteoclastic bone resorption and osteoblastic bone formation in response to mechanical stimuli. We previously developed a mathematical model of trabecular bone remodeling incorporating the possible mechanisms of cellular mechanosensing and intercellular communication in which we assumed that interstitial fluid flow activates the osteocytes to regulate bone remodeling. While the proposed model has been validated by the simulation of remodeling of a single trabecula, it remains unclear whether it can successfully represent in silico the functional adaptation of cancellous bone with its multiple trabeculae. In the present study, we demonstrated the response of cancellous bone morphology to uniaxial or bending loads using a combination of our remodeling model with the voxel finite element method. In this simulation, cancellous bone with randomly arranged trabeculae remodeled to form a well-organized architecture oriented parallel to the direction of loading, in agreement with the previous simulation results and experimental findings. These results suggested that our mathematical model for trabecular bone remodeling enables us to predict the reorganization of cancellous bone architecture from cellular activities. Furthermore, our remodeling model can represent the phenomenological law of bone transformation toward a locally uniform state of stress or strain at the trabecular level

Cell Condensation Triggers the Differentiation of Osteoblast Precursor Cells to Osteocyte-Like Cells

Though the three-dimensional (3D) in vitro culture system has received attention as a powerful tool for conducting biological research, in vitro bone formation and osteocyte differentiation studies have mostly been based on results obtained using two-dimensional (2D) culture systems. Here, we introduced a rotatory culture system to fabricate 3D spheroids, using mouse osteoblast precursor cells. These spheroids, incubated for 2 days without chemical induction by osteogenic supplements, exhibited notably up-regulated osteocyte marker levels; osteoblast marker levels were down-regulated, as compared to those of the conventional 2D monolayer model. The cell condensation achieved with the 3D spheroid structure triggered a greater level of differentiation of osteoblast precursor cells into osteocyte-like cells than that observed during chemical induction. Our study might imply that osteoblasts proliferate and become condensed at the targeted bone remodeling site, because of which osteoblasts achieved the capability to differentiate into osteocytes in vivo

Theoretical concept of cortical to cancellous bone transformation

Microstructures of cortical and cancellous bones are altered continually by load-adaptive remodeling; in addition, their cellular mechanisms are similar despite the remarkably different porosities. The cortico-cancellous transitional zone is a site of vigorous remodeling, and intracortical remodeling cavitates the inner cortex to promote its trabecularization, which is considered the main cause of bone loss because of aging. Therefore, to prevent and treat age-related cortical bone loss effectively, it is indispensable to gain an integrated understanding of the cortical to the cancellous bone transformation via remodeling. We propose a novel theoretical concept to account for the transformation of dense cortical bone to porous cancellous bone. We develop a mathematical model of cortical and cancellous bone remodeling based on the concept that bone porosity is determined by the balance between the load-bearing function of mineralized bone and the material-transporting function of bone marrow. Remodeling simulations using this mathematical model enable the reproduction of the microstructures of cortical and cancellous bones simultaneously. Furthermore, current remodeling simulations have the potential to replicate cortical-to-cancellous bone transformation based on changes in the local balance between bone formation and resorption. We anticipate that the proposed mathematical model of cortical and cancellous bone remodeling will contribute to highlighting the essential features of cortical bone loss due to trabecularization of the cortex and help predict its spatial and temporal behavior during aging

Characterization of Self-organied Osteocytic Spheroids Using Mouse Osteoblast-like Cells

Osteocyte plays a central role as a commander in the bone to modulate bone remodeling processes. While the osteocyte is known to be differentiated from osteoblasts, understanding in mechanism of the osteocyte differentiation remained still poor. The aim of this study is to elucidate the osteocyte differentiation capability using three-dimensional (3D) cell culture technique. We first fabricated a self-organized spheroid reconstructed by mouse osteoblast-like cells by adjusting the number of subcultured cells in the round-bottom well. Compared to a conventional two-dimensional (2D) monolayer model, the 3D spheroid exerted greater osteocyte gene expressions in vitro within 2 days. As a result of the size-dependent experiment, there might be an appropriate cell-cell and cell-ECM interaction for osteoblast-like cells to induce the osteocytogenesis in the form of 3D spheroid culture. Moreover, the present model showed that the spheroid further exerted the prolonged osteocyte differentiation capability after a long period of incubation, 7 days. In conclusion, we characterized the self-organized osteocytic spheroids reconstructed by osteoblast-like cells and further suggested the potential application of the spheroid as a new in vitro tissue-engineered osteocytic model

Development of continuum-based particle models of cell growth and proliferation for simulating tissue morphogenesis

Biological tissues acquire various characteristic shapes through morphogenesis. Tissue shapes result from the spatiotemporally heterogeneous cellular activities influenced by mechanical and biochemical environments. To investigate multicellular tissue morphogenesis, this study aimed to develop a novel multiscale method that can connect each cellular activity to the mechanical behaviors of the whole tissue by constructing continuum-based particle models of cellular activities. This study proposed mechanical models of cell growth and proliferation that are expressed as volume expansion and cell division by extending the material point method. By simulating cell hypertrophy and proliferation under both free and constraint conditions, the proposed models demonstrated potential for evaluating the mechanical state and tracing cells throughout tissue morphogenesis. Moreover, the effect of a cell size checkpoint was incorporated into the cell proliferation model to investigate the mechanical behaviors of the whole tissue depending on the condition of cellular activities. Consequently, the accumulation of strain energy density was suppressed because of the influence of the checkpoint. In addition, the whole tissues acquired different shapes depending on the influence of the checkpoint. Thus, the models constructed herein enabled us to investigate the change in the mechanical behaviors of the whole tissue according to each cellular activity depending on the mechanical state of the cells during morphogenesis

An energy landscape approach to understanding variety and robustness in tissue morphogenesis

During morphogenesis in development, multicellular tissues deform by mechanical forces induced by spatiotemporally regulated cellular activities, such as cell proliferation and constriction. Various morphologies are formed because of various spatiotemporal combinations and sequences of multicellular activities. Despite its potential to variations, morphogenesis is a surprisingly robust process, in which qualitatively similar morphologies are reproducibly formed even under spatiotemporal fluctuation of multicellular activities. To understand these essential characteristics of tissue morphogenesis, which involves the coexistence of various morphologies and robustness of the morphogenetic process, in this study, we propose a novel approach to capture the overall view of morphogenesis from mechanical viewpoints. This approach will enable visualization of the energy landscape, which includes morphogenetic processes induced by admissible histories of cellular activities. This approach was applied to investigate the morphogenesis of a sheet-like tissue with curvature, where it deformed to a concave or convex morphology depending on the history of growth and constriction. Qualitatively different morphologies were produced by bifurcation of the valley in the energy landscape. The depth and steepness of the valley near the stable states represented the degree of robustness to fluctuations of multicellular activities. Furthermore, as a realistic example, we showed an application of this approach to luminal folding observed in the initial stage of intestinal villus formation. This approach will be helpful to understand the mechanism of how various morphologies are formed and how tissues reproducibly achieve specific morphologies

Screening and Selection of Hypoallergenic Buckwheat Species

Both common buckwheat (Fagopyrum esculentum) flour and meal cause an allergy in sensitive patients, and if unnoticed, it can be fatal. It has become a potential occupational hazard for some mill workers. The development of hypoallergenic buckwheat would be more efficient if natural mutants for allergenic protein are detected. A screening and selection method was developed using SDS-PAGE coupled with PCR techniques. SDS-PAGE analysis of 14 different species of buckwheat revealed that F. lineare and F. urophyllum lack the 22-kDa major allergenic protein. PCR-based screening with specific primers for sequences encoding the allergenic protein was also effective in distinguishing the allergen-deficient species