Elsevier BVActa Medica Okayama8756-32822092026PPy-coated wire actuators for micromechanostimulation of cells – identification of immediate-early responsive mechanoregulatory genes in osteoblasts117914ENJiaminChenDepartment of Orthodontics, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesAmaia B.Ortega-SantosDepartment of Physics, Chemistry and Biology (IFM), Linköping UniversitySatoruHayanoDepartment of Orthodontics, Okayama University HospitalZiyiWangDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesJose G.MartinezDepartment of Physics, Chemistry and Biology (IFM), Linköping UniversityEmilio SatoshiHaraDepartment of Advanced International and Information Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesEdwin W.H.JagerDepartment of Physics, Chemistry and Biology (IFM), Linköping UniversityHiroshiKamiokaDepartment of Orthodontics, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesMechanotransduction, i.e., the conversion of mechanical cues into biochemical signals, is essential for bone development, remodeling, and adaptation. Although mechanical loading is known to regulate osteoblast function and bone homeostasis, dissecting the early and sustained mechanotransductive responses at the microscale remains challenging due to limitations of existing in vitro models. Here, we report the development and application of a mechanostimulation system comprising a polypyrrole (PPy)-based wire actuator that expands and contracts (4 m in radius) upon electrical actuation and enables precise, localized micromechanical stimulation of a small number of cells within standard culture formats. Using this system, we applied short-term (30 min) cyclic (Cyc30) or static (Stat30), as well as prolonged (120 min) cyclic (Cyc120) stimulations to two osteoblast-like cells (MC3T3-E1 or KUSA-A1). Subsequent transcriptomic profiling and computational network analyses revealed that Cyc30 was not capable of inducing significant changes in mRNA expression, suggesting cellular adaptation to short-term cyclic loading. In contrast, Stat30 induced the upregulation of Fos, Btg2, Egr1, and Fosl1, all known genes associated with mechanotransduction, supporting the validity and reproducibility of our experimental mechanostimulation system. Notably, two long non-coding RNAs (B930036N10Rik and 5430431A17Rik) were identified for the first time as being upregulated in response to Stat30 stimuli. Among the differentially expressed genes (DEGs) upregulated by Cyc120 stimuli, Hmox1, a stress-inducible enzyme known for its roles in maintaining cellular homeostasis and promoting survival, was the only DEG repeatedly observed across the Cyc30/Cyc120 and Stat30/Cyc120 comparisons in both cell types, potentially emerging as a key stress-response gene under prolonged mechanical loading. Collectively, these results establish the PPy-based microactuator as a powerful tool for microscale mechanobiology, and provide molecular insight into immediate-early responsive transcriptional programs underlying osteoblastic mechanoadaptation conserved across different cell types.No potential conflict of interest relevant to this article was reported.WileyActa Medica Okayama2688-4046632026PPy]Coated Wire Actuators for the Micromechanostimulation of Cells: Fabrication and Characterizatione202500639ENAmaia B.Ortega]SantosSensor and Actuator Systems, Department of Physics Chemistry and Biology (IFM), Linköping UniversitySatoruHayanoDepartment of Orthodontics, Okayama University HospitalEmilio SatoshiHaraAdvanced Research Center for Oral and Craniofacial Sciences Dental School, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesJose G.MartínezSensor and Actuator Systems, Department of Physics Chemistry and Biology (IFM), Linköping UniversityHiroshiKamiokaDepartment of Orthodontics, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesEdwin W. H.JagerSensor and Actuator Systems, Department of Physics Chemistry and Biology (IFM), Linköping UniversityCellular mechanotransduction signals play a crucial role in physiological and pathological conditions, including skeletal disorders. Although various systems exist to mechanically stimulate cultured cells, most are constrained by incubator incompatibility, limited physiological relevance, nonuniform stimulation, or complexity. The objective of this article is to develop and validate a compact, incubator-compatible tool capable of delivering localized and physiologically relevant mechanical stimulation to small cell populations. Here, we introduce a polypyrrole-based wire-shaped microactuator designed to induce localized mechanical stress to adjacent cells. These wire-shaped microactuators are biocompatible, easy-to-use, and compact for use within standard in vitro cell culture systems. Using a noncontact optical method, we characterize the actuation of polypyrrole-coated wires in an aqueous NaDBS electrolyte, showing radial expansion of 1.5–8 µm depending on the deposited polypyrrole film thickness, comparable to cellular dimensions. Next, the actuation is confirmed to be robust and stable to use in cell culture media at physiological temperature. To evaluate biological relevance, osteoblastic KUSA-A1 cells are mechanically stimulated inside the incubator and transcriptomic changes are assessed. Mechanical stimulation resulted in upregulation of genes previously associated with mechanotransduction, including Fos and Fosb. Additionally, several uncharacterized long noncoding RNAs are differentially expressed, suggesting potential novel players in the mechanotransduction pathway.No potential conflict of interest relevant to this article was reported.Ovid Technologies (Wolters Kluwer Health)Acta Medica Okayama2169-75741422026A Technique for Repositioning the Posteriorly Displaced Premaxilla Following Prior Repair of Complete Bilateral Cleft Lipe7467ENYukiArimuraDepartment of Oral and Maxillofacial Reconstructive Surgery, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical SciencesSeijiIidaDepartment of Oral and Maxillofacial Reconstructive Surgery, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical SciencesAikoHyodoAdvanced Cleft Lip and Cleft Palate Center, Okayama University HospitalAyakaMikamiDepartment of Oral and Maxillofacial Reconstructive Surgery, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical SciencesSatoruHayanoAdvanced Cleft Lip and Cleft Palate Center, Okayama University HospitalFumikoTakemotoAdvanced Cleft Lip and Cleft Palate Center, Okayama University HospitalHiroshiKamiokaAdvanced Cleft Lip and Cleft Palate Center, Okayama University HospitalIt is well known that osteotomy of the premaxilla is an effective surgical procedure for the correction of a displaced premaxilla in patients with bilateral cleft lip and palate. In cases with a posteriorly displaced premaxilla, it is not easy to move the premaxilla forward because of scarring of the palatal mucosal attachment, narrowing of the adjacent maxillary segments, and the stable fixation of this bone segment after its movement. This fixation is also important in cases without secondary bone grafting. We propose a new method that combines osteotomy and a method such as bone distraction for cases with significant premaxilla displacement that are difficult to repair by osteotomy alone. A conventional orthodontic palatal expander was used as the distractor. The anterior arms were bent at the posterior part of the lingual side of the anterior teeth, and a resin base was attached to the arm parts. The posterior arms were bent and waxed onto the bands of both first molars. Supportive stainless steel wire arms, which are attached to the rest of the deciduous molars, stabilize the distractor. After the osteotomy of the premaxilla, distraction was performed at a rate of 1.0 mm per day, starting the day after surgery. Because the premaxilla of patients with bilateral cleft lip and palate has undergone multiple surgical interventions, the soft tissue is not mobile, making it impossible to guide the premaxilla to an ideal position in a single stage. However, this procedure, using this semirigid distractor, makes it possible to move the osteotomized premaxilla to the planned position with firm stability.No potential conflict of interest relevant to this article was reported.MDPI AGActa Medica Okayama2221-37591412026The Influence of Fluidic Flow Stress on the Development of the Secondary Palate9ENMasayoNagataDepartment of Orthodontics, Okayama University HospitalSatoruHayanoDepartment of Orthodontics, Okayama University HospitalZiyiWangDepartment of Molecular Biology and Biochemistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityTakahiroKosamiDepartment of Orthodontics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityHiroshiKamiokaDepartment of Orthodontics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityCraniofacial development is orchestrated by a finely regulated interplay of numerous genes and signaling pathways. Palatogenesis proceeds through a complex, stepwise process, in which endogenous mechanical stresses within tissues have been implicated. However, the impact of exogenous fluidic flow mechanical stress derived from maternal movement on palatal development remains unclear. In this study, we investigated the effect of exogenous fluidic flow mechanical stress on palatal morphogenesis, focusing on the horizontal outgrowth of palatal shelves after elevation. Palatal tissues dissected from mouse embryos were subjected to organ culture with or without mechanical loading (loaded and unloaded groups, respectively). Stress magnitude was quantified by calculating wave energy, and morphometric and molecular analyses were performed. Compared with the unloaded group, palatal shelves in the loaded group showed significant increases in thickness and volume, accompanied by enhanced cell proliferation, nuclear translocation of YAP and -catenin, and upregulation of the osteogenic markers Osterix and Osteocalcin. No significant difference in apoptosis was observed. These findings indicate that exogenous mechanical stress promotes cell proliferation and osteogenic differentiation through the Hippo and WNT/-catenin pathways in palate explants. Our results suggest that moderate maternal movement-induced mechanical stress contributes to normal palatogenesis, providing new insights into the mechanisms underlying cleft palate.No potential conflict of interest relevant to this article was reported.Elsevier BVActa Medica Okayama0003-99691552023Ruxolitinib altered IFN- induced necroptosis of human dental pulp stem cells during osteoblast differentiation105797ENAtsukoTanakaDepartment of Orthodontics, Okayama University HospitalSatoruHayanoDepartment of Orthodontics, Okayama University HospitalMasayoNagataDepartment of Orthodontics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityTakahiroKosamiDepartment of Orthodontics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityZiyiWangDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesHiroshiKamiokaDepartment of Orthodontics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityObjective: This study aimed to evaluate the role of ruxolitinib in the interferon beta (IFN-) mediated osteoblast differentiation using human dental pulp stem cells (hDPSCs).<br>
Design: hDPSCs from five deciduous teeth of healthy patients were stimulated by adding human recombinant IFN- 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.<br>
Results: In the IFN--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--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.<br>
Conclusion: Ruxolitinib interfered with the IFN--mediated necroptosis and osteogenic differentiation via the JAK-STAT pathway.No potential conflict of interest relevant to this article was reported.WileyActa Medica Okayama1320-546320212021Investigation of the molecular causes underlying physical abnormalities in Diamond]Blackfan anemia patients with RPL5 haploinsufficiency111ENYukoFukuiDepartment of Orthodontics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Okayama University SatoruHayanoDepartment of Orthodontics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Okayama UniversityNoriakiKawanabeDepartment of Orthodontics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Okayama UniversityZiyiWangDepartment of Orthodontics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Okayama UniversityAkiraShimadaDepartment of Pediatric Hematology/Oncology Okayama University Hospital Megumu K.SaitoDepartment of Clinical Application, Center for iPS Cell Research and Application Kyoto UniversityIsaoAsakaDepartment of Fundamental Cell Technology, Center for iPS Cell Research and Application Kyoto UniversityHiroshiKamiokaDepartment of Orthodontics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Okayama UniversityDiamond-Blackfan anemia (DBA) is a genetic disorder caused by mutations in genes encoding ribosomal proteins and characterized by erythroid aplasia and various physical abnormalities. Although accumulating evidence suggests that defective ribosome biogenesis leads to p53-mediated apoptosis in erythroid progenitor cells, little is known regarding the underlying causes of the physical abnormalities. In this study, we established induced pluripotent stem cells from a DBA patient with RPL5 haploinsufficiency. These cells retained the ability to differentiate into osteoblasts and chondrocytes. However, RPL5 haploinsufficiency impaired the production of mucins and increased apoptosis in differentiated chondrocytes. Increased expression of the pro-apoptotic genes BAX and CASP9 further indicated that RPL5 haploinsufficiency triggered p53-mediated apoptosis in chondrocytes. MDM2, the primary negative regulator of p53, plays a crucial role in erythroid aplasia in DBA patient. We found the phosphorylation level of MDM2 was significantly decreased in RPL5 haploinsufficient chondrocytes. In stark contrast, we found no evidence that RPL5 haploinsufficiency impaired osteogenesis. Collectively, our data support a model in which RPL5 haploinsufficiency specifically induces p53-mediated apoptosis in chondrocytes through MDM2 inhibition, which leads to physical abnormalities in DBA patients.No potential conflict of interest relevant to this article was reported.