Ruxolitinib altered IFN-β induced necroptosis of human dental pulp stem cells during osteoblast differentiation

Objective: This study aimed to evaluate the role of ruxolitinib in the interferon beta (IFN-β) mediated osteoblast differentiation using human dental pulp stem cells (hDPSCs). Design: hDPSCs from five deciduous teeth of healthy patients were stimulated by adding human recombinant IFN-β protein (1 or 2 ng/ml) to the osteogenic differentiation induction medium. Substrate formation was determined using Alizarin Red staining, calcium concentration, and osteoblast marker expression levels. Ruxolitinib was used to inhibit the Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathway. Apoptosis was detected using terminal deoxynucleotidyl nick-end labeling (TUNEL) staining, and necroptosis was detected using propidium iodide staining and phosphorylated mixed lineage kinase domain-like protein (pMLKL) expression. Results: In the IFN-β-treated group, substrate formation was inhibited by a reduction in alkaline phosphatase (ALP) expression in a concentration-dependent manner. Although the proliferation potency was unchanged between the IFN-β-treated and control groups, the cell number was significantly reduced in the experimental group. TUNEL-positive cell number was not significantly different; however, the protein level of necroptosis markers, interleukin-6 (IL-6) and pMLKL were significantly increased in the substrate formation. Cell number and ALP expression level were improved in the group administered ruxolitinib, a JAK-STAT inhibitor. Additionally, ruxolitinib significantly suppressed IL-6 and pMLKL levels. Conclusion: Ruxolitinib interfered with the IFN-β-mediated necroptosis and osteogenic differentiation via the JAK-STAT pathway

Strain uses gap junctions to reverse stimulation of osteoblast proliferation by osteocytes

Identifying mechanisms by which cells of the osteoblastic lineage communicate in vivo is complicated by the mineralised matrix that encases osteocytes, and thus, vital mechanoadaptive processes used to achieve load‐bearing integrity remain unresolved. We have used the coculture of immunomagnetically purified osteocytes and primary osteoblasts from both embryonic chick long bone and calvariae to examine these mechanisms. We exploited the fact that purified osteocytes are postmitotic to examine both their effect on proliferation of primary osteoblasts and the role of gap junctions in such communication. We found that chick long bone osteocytes significantly increased basal proliferation of primary osteoblasts derived from an identical source (tibiotarsi). Using a gap junction inhibitor, 18β‐glycyrrhetinic acid, we also demonstrated that this osteocyte‐related increase in osteoblast proliferation was not reliant on functional gap junctions. In contrast, osteocytes purified from calvarial bone failed to modify basal proliferation of primary osteoblast, but long bone osteocytes preserved their proproliferative action upon calvarial‐derived primary osteoblasts. We also showed that coincubated purified osteocytes exerted a marked inhibitory action on mechanical strain–related increases in proliferation of primary osteoblasts and that this action was abrogated in the presence of a gap junction inhibitor. These data reveal regulatory differences between purified osteocytes derived from functionally distinct bones and provide evidence for 2 mechanisms by which purified osteocytes communicate with primary osteoblasts to coordinate their activity

The Amazing Osteocyte

The last decade has provided a virtual explosion of data on the molecular biology and function of osteocytes. Far from being the “passive placeholder in bone,” this cell has been found to have numerous functions, such as acting as an orchestrator of bone remodeling through regulation of both osteoclast and osteoblast activity and also functioning as an endocrine cell. The osteocyte is a source of soluble factors not only to target cells on the bone surface but also to target distant organs, such as kidney, muscle, and other tissues. This cell plays a role in both phosphate metabolism and calcium availability and can remodel its perilacunar matrix. Osteocytes compose 90% to 95% of all bone cells in adult bone and are the longest lived bone cell, up to decades within their mineralized environment. As we age, these cells die, leaving behind empty lacunae that frequently micropetrose. In aged bone such as osteonecrotic bone, empty lacunae are associated with reduced remodeling. Inflammatory factors such as tumor necrosis factor and glucocorticoids used to treat inflammatory disease induce osteocyte cell death, but by different mechanisms with potentially different outcomes. Therefore, healthy, viable osteocytes are necessary for proper functionality of bone and other organs. © 2011 American Society for Bone and Mineral Research

Fluid flow in the osteocyte mechanical environment : a fluid-structure interaction approach

Osteocytes are believed to be the primary sensor of mechanical stimuli in bone, which orchestrate osteoblasts and osteoclasts to adapt bone structure and composition to meet physiological loading demands. Experimental studies to quantify the mechanical environment surrounding bone cells are challenging, and as such, computational and theoretical approaches have modelled either the solid or fluid environment of osteocytes to predict how these cells are stimulated in vivo. Osteocytes are an elastic cellular structure that deforms in response to the external fluid flow imposed by mechanical loading. This represents a most challenging multi-physics problem in which fluid and solid domains interact, and as such, no previous study has accounted for this complex behaviour. The objective of this study is to employ fluid–structure interaction (FSI) modelling to investigate the complex mechanical environment of osteocytes in vivo. Fluorescent staining of osteocytes was performed in order to visualise their native environment and develop geometrically accurate models of the osteocyte in vivo. By simulating loading levels representative of vigorous physiological activity (3,000με compression and 300 Pa pressure gradient), we predict average interstitial fluid velocities (∼60.5μ m/s ) and average maximum shear stresses (∼11 Pa ) surrounding osteocytes in vivo. Interestingly, these values occur in the canaliculi around the osteocyte cell processes and are within the range of stimuli known to stimulate osteogenic responses by osteoblastic cells in vitro. Significantly our results suggest that the greatest mechanical stimulation of the osteocyte occurs in the cell processes, which, cell culture studies have indicated, is the most mechanosensitive area of the cell. These are the first computational FSI models to simulate the complex multi-physics mechanical environment of osteocyte in vivo and provide a deeper understanding of bone mechanobiology

Cytoskeletal Dynamics: A View from the Membrane

Many aspects of cytoskeletal assembly and dynamics can be recapitulated in vitro; yet, how the cytoskeleton integrates signals in vivo across cellular membranes is far less understood. Recent work has demonstrated that the membrane alone, or through membrane-associated proteins, can effect dynamic changes to the cytoskeleton, thereby impacting cell physiology. Having identified mechanistic links between membranes and the actin, microtubule, and septin cytoskeletons, these studies highlight the membrane’s central role in coordinating these cytoskeletal systems to carry out essential processes, such as endocytosis, spindle positioning, and cellular compartmentalization

オウヨウセイブツカガクブ1ネンセイ ヲ タイショウ トシタ ショクイクトライアル ノ ジッシ ト ヒョウカ ジュギョウサンカシャ ヘノ アンケート ニヨル ケントウ

応用生物科学部1年生を対象に,食育トライアル授業として,食の生産,安全管理,健康管理などに関する10回の講義と2回の農業体験(種まき,収穫作業等)を実施し,学生の授業への出席状況及び授業前後のアンケートによって,プログラムの内容および学生の食育に対する知識,意欲等の変化を評価した。応用生物科学部全学科の学生に授業への参加を呼びかけた結果,授業の出席者は27名であり,栄養科学科の学生が20名と最も多く,参加学生の学科構成に偏りがみられた。授業の初回に行ったアンケートの回答者は24名,最終回に行ったアンケートの回答者は11名であった。授業後のアンケートでは,「食育活動への興味は深まったか」「知りたい情報は得られたか」「参加後,農業と食の関連に対する考えが変わったか」「授業として開講された場合受講するか」という問いに対し,それぞれ73～91%が「はい」と回答し,「授業への総合的な満足度」についても73%が「(非常に)満足である」と回答した。「農業」に原点をおいた食育トライアル授業によって,食べ物の成長を通した気づきと感動,生産者への感謝の気持ち,環境への配慮,栄養と健康との関連知識の習得等に寄与することができたと考えられる。今後は授業への参加者数を増やし,結果の一般化可能性について検討するとともに,授業の実現に向けた人的・物的資源の問題について検討する必要がある。We aimed to evaluate changes in both knowledge of dietary education (Shokuiku) and in motivation, in first-year students of the Faculty of Applied Biosciences who attended a voluntary Shokuiku program in 2008. The program comprised 10 lectures on food production, safety management, and health management, as well as 2 farming practicum classes (seed sowing, harvesting, etc.). Twenty of the 27 students who attended the courses belonged to the Department of Nutritional Sciences. We received responses from 24 and 11 students to questionnaires administered before and after the program, respectively. We obtained a "yes" answer 73-91% of the time in response to the following questions : 1) Was your interest in dietary education activities enhanced? 2) Were you able to obtain the information you sought? 3) After attending these courses, did your understanding of the relationship between farming and diet change? and 4) Would you take this course if it were officially offered as a class? Seventy-three percent of students replied that they were very satisfied with the courses. By placing "farming" as the central principle, these courses contributed to food awareness and appreciation, feelings of gratitude toward producers, consideration for the environment, and knowledge acquisition about the relationship between nutrition and health. Future studies should address the generalizability by examining a larger pool of participants. In addition, other issues such as human and material resources should be considered in realization of the Shokuiku program curriculum

In vitro Models of Bone Remodelling and Associated Disorders

Disruption of bone remodelling by diseases such as osteoporosis results in an imbalance between bone formation by osteoblasts and resorption by osteoclasts. Research into these metabolic bone disorders is primarily performed in vivo; however, in the last decade there has been increased interest in generating in vitro models that can reduce or replace our reliance on animal testing. With recent advances in biomaterials and tissue engineering the feasibility of laboratory-based alternatives is growing; however, to date there are no established in vitro models of bone remodelling. In vivo, remodelling is performed by organised packets of osteoblasts and osteoclasts called bone multicellular units (BMUs). The key determinant of whether osteoclasts form and remodelling occurs is the ratio between RANKL, a cytokine which stimulates osteoclastogenesis, and OPG, its inhibitor. This review initially details the different circumstances, conditions, and factors which have been found to modulate the RANKL:OPG ratio, and fundamental factors to be considered if a robust in vitro model is to be developed. Following this, an examination of what has been achieved thus far in replicating remodelling in vitro using three-dimensional co-cultures is performed, before overviewing how such systems are already being utilised in the study of associated diseases, such as metastatic cancer and dental disorders. Finally, a discussion of the most important considerations to be incorporated going forward is presented. This details the need for the use of cells capable of endogenously producing the required cytokines, application of mechanical stimulation, and the presence of appropriate hormones in order to produce a robust model of bone remodelling

Calcium response in single osteocytes to locally applied mechanical stimulus: Differences in cell process and cell body

It is proposed that osteocytes embedded in the bone matrix have the ability to sense deformation and/or damage to the matrix and to feed these mechanical signals back to the adaptive bone remodeling process. When osteoblasts differentiate into osteocytes during the bone formation process, they change their morphology to a stellate form with many slender processes. This characteristic cell shape may underlie the differences in mechanosensitivity between the cell processes and cell body. To elucidate the mechanism of cellular response to mechanical stimulus in osteocytes, we investigated the site-dependent response to quantitatively controlled local mechanical stimulus in single osteocytes isolated from chick embryos, using the technique of calcium imaging. A mechanical stimulus was applied to a single osteocyte using a glass microneedle targeting a microparticle adhered to the cell membrane by modification with a monoclonal antibody OB7.3. Application of the local deformation induced calcium transients in the vicinity of the stimulated point and caused diffusive wave propagation of the calcium transient to the entire intracellular region. The rate of cell response to the stimulus was higher when applied to the cell processes than when applied to the cell body. In addition, a large deformation was necessary at the cell body to induce calcium transients, whereas a relatively small deformation was sufficient at the cell processes, suggesting that the mechanosensitivity of the cell processes was higher than that of the cell body. These results suggest that the cell shape with slender processes contributes to the site-dependent mechanosensitivity in osteocytes