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Extracellular vesicles (EVs) are natural carriers for intercellular communication within the human body. Mimicking and utilizing EVs by combining them with artificial nanocarriers such as liposomes for drug delivery has garnered considerable attention. However, current technologies for manipulating EVs to facilitate their fusion with liposomes are limited; the existing technique of polyethylene glycol (PEG)-induced fusion is highly inefficient for fusion. In our previous study, we demonstrated that membrane fusion could be induced by Tat peptide (YGRKKRRQRRR)-conjugated poly(ethylene glycol)-phospholipids (Tat-PEG-lipids), in which the Tat peptide and lipid domain facilitate membrane attachment and subsequent fusion between cells and liposomes. This approach is promising for forming EV and liposomal hybrids. In this study, we aim to fuse EVs and liposomes using Tat-PEG-lipids. We isolated and characterized EVs derived from HEK293T cell culture medium and treated a mixture of EVs and liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and cholesterol (1:1, molar ratio), with Tat-PEG-lipids with different lipid chain lengths. Here, we used nonanoyl (C9), dodecanoyl (C12), and myristoyl (C14) groups as lipid anchors with 5 kDa PEG chains. Dynamic light scattering analysis revealed a large increase in the apparent size of mixture of EVs and liposomes by adding Tat-PEG-lipids (especially C14, C12, followed by C9). Fluorescence resonance energy transfer, confocal laser scanning microscopy, and transmission electron microscopy, used to analyze the reaction process, revealed that the membrane fusion occurred between EVs and liposomes but not their aggregates. The short lipid domain of Tat-PEG-lipids effectively induced membrane fusion and the formation of hybrid EVs and liposomes. Thus, Tat-PEG-lipids (C9 and C12) could be promising candidates for inducing membrane fusion to fabricate EV-liposome hybrids.

With the rising prevalence of bone-related injuries, it is crucial to improve treatments for fractures and defects. Tissue engineering offers a promising solution in the form of injectable hydrogel scaffolds that can sustain the release of growth factors like bone morphogenetic protein-2 (BMP-2) for bone repair. Recently, we discovered that tetra-PEG hydrogels (Tetra gels) undergo gel-gel phase separation (GGPS) at low polymer content, resulting in hydrophobicity and tissue affinity. In this work, we examined the potential of a newer class of gel, the oligo-tetra-PEG gel (Oligo gel), as a growth factor-releasing scaffold. We investigated the extent of GGPS occurring in the two gels and assessed their ability to sustain BMP-2 release and osteogenic potential in a mouse calvarial defect model. The Oligo gel underwent a greater degree of GGPS than the Tetra gel, exhibiting higher turbidity, hydrophobicity, and pore formation. The Oligo gel demonstrated sustained protein or growth factor release over a 21-day period from protein release kinetics and osteogenic cell differentiation studies. Finally, BMP-2-loaded Oligo gels achieved complete regeneration of critical-sized calvarial defects within 28 days, significantly outperforming Tetra gels. The easy formulation, injectability, and capacity for sustained release makes the Oligo gel a promising candidate therapeutic biomaterial.

Vertebrates form their skeletal tissues from three distinct origins (the neural crest, paraxial mesoderm, and lateral plate mesoderm) through two distinct modes of ossification (intramembranous and endochondral ossification). Since the paraxial mesoderm generates both intramembranous and endochondral bones, it is thought to give rise to both osteoprogenitors and osteo-chondroprogenitors. However, it remains unclear what directs the paraxial mesoderm-derived cells toward these different fates in distinct skeletal elements during human skeletal development. To answer this question, we need experimental systems that recapitulate paraxial mesoderm-mediated intramembranous and endochondral ossification processes. In this study, we aimed to develop a human pluripotent stem cell (hPSC)-based system that models the human intramembranous ossification process. We found that spheroid culture of the hPSC-derived paraxial mesoderm derivatives generates osteoprogenitors or osteo-chondroprogenitors depending on stimuli. The former induced intramembranous ossification, and the latter endochondral ossification, in mouse renal capsules. Transcriptional profiling supported the notion that bone signatures were enriched in the intramembranous bone-like tissues. Thus, we developed a system that recapitulates intramembranous ossification, and that enables the induction of two distinct modes of ossification by controlling the cell fate of the hPSC-derived paraxial mesoderm derivatives.

Abstract Liquid–liquid phase separation (LLPS), an important phenomenon in the field of polymer science and material design, plays an essential role in cells and living bodies. Poly(vinyl alcohol) (PVA) is a popular semicrystalline polymer utilized in the synthesis of artificial biomaterials. The aqueous solutions of its derivatives with tuned degrees of saponification (DS) exhibit LLPS. However, the miscibility and LLPS behavior of PVA aqueous solution are still unclear. This study describes the miscibility diagram of the ternary mixture, where water and two types of poly(vinyl alcohol) (PVA) with different DSs [98 (PVA98), 88 (PVA88), 82 (PVA82), and 74 mol% (PVA74)] were blended. UV–Vis measurement was conducted to evaluate the miscibility. Immiscibility was more pronounced at elevated temperatures, exhibiting LLPS. The ternary immiscibility diagram, displaying miscible–immiscible behaviors in the aqueous mixtures of PVA74:PVA98, PVA82:PVA98, and PVA88:PVA98 (blended at a constant volume ratio), indicated that increasing the concentration, temperature, and blend ratio of PVAs at a lower DS increased immiscibility, suggesting that the free energy of mixing increases with increasing these parameters. The miscible–immiscible behaviors of PVAs/water systems provide fundamental knowledge about LLPS and the design of PVA-based materials.

Exosomes (diameter 30–200 nm) are a subtype of extracellular vesicles secreted by cells containing DNA, microRNA (miRNA), and proteins. Exosomes are expected to be valuable as a means of delivering drugs or functional miRNAs in treatment of diseases. However, the delivery of exosomes is not sufficiently effective, even though exosomes have intrinsic delivery functions. Cell-penetrating peptides (CPPs) are short peptide families that facilitate cellular intake of molecules and vesicles. We previously reported that the modification of cells, and liposomes with CPP-conjugated-lipids, CPPs conjugated with poly (ethylene glycol)-conjugated phospholipids (PEG-lipid), that induce adhesion by CPPs, can be useful for cell-based assays and harvesting liposomes. In this study, we aimed to modulate the exosome surface using Tat peptide (YGRKKRRQRRR)-PEG-lipids to improve intracellular delivery to endothelial cells. We isolated and characterized exosomes from the medium of HEK 293 T cell cultures. Tat conjugated PEG-lipids with different spacer molecular weights and lipid types were incorporated into exosomes using fluorescein isothiocyanate labeling to optimize the number of Tat-PEG-lipids immobilized on the exosome surface. The exosomes modified with Tat-PEG-lipids were incubated with human umbilical vein endothelial cells (HUVECs) to study the interaction. Tat conjugated with 5 kDa PEG and C16 lipids incorporated on the exosome surface were highly detected inside HUVECs by flow cytometry. Fluorescence was negligible in HUVECs for control groups. Thus, Tat-PEG-lipids can be modified on the exosome surface, improving the intracellular delivery of exosomes.

Introduction: Aging, genetic mutations, and other pathological conditions cause impairment of skeletal growth and bone metabolism, which affect activities of daily living and quality of life in all life stages. Although several drugs have been used in clinical settings and new drugs have been developed for the treatment of skeletal degenerative disorders such as osteoporosis and genetic disorders such as osteogenesis imperfecta (OI), there is clear demand for development of new drugs, especially orally available anabolic drugs that are applicable for a wide range of skeletal disorders. Methods: To identify therapeutic candidates for skeletal disorders, peptide screening was performed. To validate the identified peptides, we performed a bone histomorphometric analysis with rat bone tissues and in vitro cell proliferation assays of skeletal cells. To understand the metabolism of the peptides, we performed a biochemical analysis, followed by in vitro assays for proliferation and differentiation of skeletal cells. We examined the therapeutic efficacy of the identified peptides with several mouse models representing skeletal disorders including bone fracture, osteoporosis, and osteogenesis imperfecta. In vivo therapeutic effects of the candidate were assessed with radiological analysis and mechanical property tests. Results: We identified the egg yolk-derived functional peptide PF201. PF201 promoted in vivo bone formation in rodents and enhanced proliferation of osteoblasts and chondrocytes in vitro. D2, a metabolite of PF201, was present and circulated after digestion and absorption in the digestive tract. D2 had positive impacts on the proliferation and differentiation of mesenchymal stem cells and preosteoblasts. Oral administration of D2 accelerated bone healing in a mouse fracture model. D2 also improved bone strength and fracture healing under ovariectomy-induced osteoporotic conditions in mice, and D2 showed a therapeutic effect in a mouse OI model. Conclusion: D2 is likely to be a candidate for an orally available therapeutic for a range of skeletal disorders.

Introduction: Activation of skeletal progenitors upon tissue injury and the subsequent cell fate specification are tightly coordinated in the bone repair process. Although known osteoimmunological signaling networks play important roles in the microenvironment of the bone defect sites, the molecular mechanism underlying the bone repair process has not been fully understood. Methods: To better understand the behavior of the skeletal progenitors and the heterogeneity of the cells during bone repair at the microenvironmental level, we performed a combinatorial analysis consisting of lineage tracing for skeletal progenitors using the Sox9-CreERT2;R26RtdTomato mouse line followed by single-cell RNA sequencing (scRNA-seq) analysis using a mouse model of calvarial bone repair. To identify a therapeutic target for bone regeneration, further computational analysis was performed focusing on the identification of the cell–cell interactions, followed by pharmacological assessments with a critical-size calvarial bone defect mouse model. Results: Lineage tracing analysis showed that skeletal progenitors marked by Sox9 were activated upon bone injury and contributed to bone repair by differentiating into osteoblasts. The scRNA-seq analysis characterized heterogeneous cell populations at the bone defect sites; the computational analysis predicted a bifurcated lineage from skeletal progenitors toward osteogenic and adipogenic lineages. Chemokine C–C motif ligand 9 (Ccl9) was identified as a signaling molecule that regulates bone regeneration in the mouse model, possibly through the regulation of adipogenic differentiation at the bone defect site. Conclusion: Multipotential skeletal progenitors and the direction of the cell differentiation were characterized at single cell resolution in a mouse bone repair model. The Ccl9 signaling pathway may be a key factor directing osteogenesis from the progenitors in the model and may be a therapeutic target for bone regeneration.

Possible distinct contributions of Runx 2 and Runx3 in osteoarthritis have not been clarified. Nagata et al. show that Runx3 protects adult articular cartilage by extracellular matrix protein production in normal conditions, while Runx2 exerts both catabolic and anabolic effects during inflammation.

Quantifying health status and identifying modifiable factors are essential for effective and individualized prevention of age-related conditions and for promoting health during aging. The ME-BYO concept from Kanagawa Prefecture, one of Japan’s largest prefectures, can be used to establish a healthy aging society. In disease etiology, ME-BYO considers the status of an individual’s body and mind as changing continuously from healthy to sick instead of being a dichotomy between the two. ME-BYO conceptualizes the entire process of this change. The ME-BYO index was developed in 2019 to comprehensively and numerically measure and visualize an individual’s current health status and future disease risk by quantifying data on the four domains of metabolic function, locomotor function, cognitive function, and mental resilience. The ME-BYO index has been implemented in the personal health management application “My ME-BYO.” However, scientific validation of this index and the development of a practical application using healthcare data remain to be completed. In 2020, our research team started a project to refine the ME-BYO index using data from the Kanagawa ME-BYO prospective cohort study, which is a large population-based genomic cohort study. This project will scientifically evaluate the ME-BYO index and develop a practical application for promoting healthy aging.

Abstract Objective Cognitive decline is an important and well-documented health problem. The Mini-Cog, a simple cognitive function test, is recommended as a potential early cognitive screening tool. Kanagawa Prefecture, one of the largest prefectures in Japan, developed this self-testing application on a smartphone to enable a large number of residents to assess their cognitive function. This study aimed to verify the validity and reliability of the Mini-Cog. Results Twenty men and 20 women aged 65–85 years were enrolled. Criterion-related validity of the method tested by professional staff was found to have an acceptable positive association. The test–retest reliability was lower than the clinically expected intraclass correlation coefficient value because of the inclusion of learning and order effects. If the Mini-Cog score of this application is low, the system is equipped with a function that advises the users on preventing cognitive decline, directing them to the appropriate services, and recommending visits to a medical institution. Therefore, the system can be used continuously as a tool for health behaviors and promotions.

The transcription factors, Runx1 and Runx2 are critical embryonically for generation of HSCs and osteoblasts, respectively. Here the authors show that adult mice lacking Runx1 and Runx2 in HSC-supporting CAR cells displayed an increase in fibrosis with reduced HSCs in bone marrow.

Hydrogels, which have polymer networks through supramolecular and reversible interactions, exhibit various mechanical responsibilities to its surroundings. The influence of the reversible bonds on a hydrogel’s macroscopic properties, such as viscoelasticity and dynamics, is not fully understood, preventing further innovative material development. To understand the relationships between the mechanical properties and molecular structures, it is required to clarify the molecular understanding of the networks solely crosslinked by reversible interactions, termed “transient networks”. This review introduces our recent progress on the studies on the molecular mechanism of viscoelasticity in transient networks using multiple methods and model materials. Based on the combination of the viscoelasticity and diffusion measurements, the viscoelastic relaxation of transient networks does not undergo the diffusion of polymers, which is not explained by the framework of conventional molecular models for the viscoelasticity of polymers. Then, we show the results of the comparison between the viscoelastic relaxation and binding dynamics of reversible bonds. Viscoelastic relaxation is primarily affected by “dissociation dynamics of the bonds” and “network structures”. These results are explained in the framework that the backbone, which is composed of essential chains supporting the stress, is broken by multiple dissociation events. This understanding of molecular dynamics in viscoelasticity will provide the foundation for designing transient networks.

ObjectiveMaintaining or improving regular walking speed can help extend healthy life expectancy and prevent frailty. The evaluation of walking speed can help individuals meet their daily exercise goals; therefore, it may be beneficial as a public health policy for residents to measure and evaluate their walking speed easily. This study aimed to verify the validity and reliability of a smartphone application for the 10-m walk test, measured in the general population.MethodsThe study participants were men (n = 20) and women (n = 20) aged 65–85 years. The 10-m walk tests were performed at the usual walking speed, using the stopwatch function of a newly developed smartphone application. A total of three 10-m walk tests were performed simultaneously with the study participants and professional fitness instructors to evaluate the criterion-related validity and the test-retest reliability.ResultsA strong positive correlation was found in the criterion-related validity by the study participants and professional staff for the average of the three trials {r = 0.961 [95% confidence interval (CI) = 0.927, 0.979]}. The results revealed good reliability, with an intraclass correlation coefficient of 0.712 (95% CI = 0.571, 0.823).ConclusionThe smartphone application walking speed measurement method can be widely used by the general public and is useful for health promotion.

OBJECTIVES: Although higher occupational classes have been reported to be associated with better health, researchers do not fully understand whether such associations derive from the position or individual characteristics of the person in that position. We examined the association between being a manager and cardiovascular disease (CVD) risk factors using unique panel data in Japan that annually observed employees’ occupational class and health conditions. METHODS: We analyzed data for 45 888 observations from a Japanese company from 2013 through 2017. The association between being a manager and CVD risk factors (metabolic risks and health-related behaviors) were evaluated using simple pooled cross-sectional analyses with adjustment for age, sex, marital status, and overtime-working hours. We further incorporated employee-level fixed-effects into the models to examine whether the associations were subject to individual time-invariant factors. RESULTS: The pooled cross-sectional analyses showed that, compared to non-managers, managers had 2.0 mg/dl lower low density lipoprotein cholesterol (LDL-C) level, 1.4 mmHg-lower systolic blood pressure, and 0.2 kg/m^2 lower body mass index (BMI). After adjusting for employee-level fixed-effects, being a manager was associated with a significantly 2.2 mg/dl higher LDL-C level. However, the associations between an individual’s management status and blood pressure or BMI were not significant. Furthermore, managers were 5.5% less likely to exercise regularly and 6.1% less likely to report sufficient sleep in the fixed-effects models, although the pooled cross-sectional analyses did not demonstrate these significant associations. CONCLUSIONS: Our findings suggest the necessity of considering these unfavorable health risks associated with being promoted to a manager.

Cardiovascular diseases (CVDs) remain a leading cause of death globally. According to the American Heart Association, approximately 19.1 million deaths were attributed to CVDs in 2020, in particular, ischemic heart disease and stroke. Several known risk factors for CVDs include smoking, alcohol consumption, lack of regular physical activity, and diabetes. The last decade has been characterized by widespread diffusion in the use of wristband-style wearable devices which can monitor and collect heart rate data, among other information. Wearable devices allow the analysis and interpretation of physiological and activity data obtained from the wearer and can therefore be used to monitor and prevent potential CVDs. However, these data are often provided in a manner that does not allow the general user to immediately comprehend possible health risks, and often require further analytics to draw meaningful conclusions. In this paper, we propose a disentangled variational autoencoder (β-VAE) with a bidirectional long short-term memory network (BiLSTM) backend to detect in an unsupervised manner anomalies in heart rate data collected during sleep time with a wearable device from eight heterogeneous participants. Testing was performed on the mean heart rate sampled both at 30 s and 1 min intervals. We compared the performance of our model with other well-known anomaly detection algorithms, and we found that our model outperformed them in almost all considered scenarios and for all considered participants. We also suggest that wearable devices may benefit from the integration of anomaly detection algorithms, in an effort to provide users more processed and straightforward information.

Abstract Human beings have adaptively rational cognitive biases for efficiently acquiring concepts from small-sized datasets. With such inductive biases, humans can generalize concepts by learning a small number of samples. By incorporating human cognitive biases into learning vector quantization (LVQ), a prototype-based online machine learning method, we developed self-incremental LVQ (SILVQ) methods that can be easily interpreted. We first describe a method to automatically adjust the learning rate that incorporates human cognitive biases. Second, SILVQ, which self-increases the prototypes based on the method for automatically adjusting the learning rate, is described. The performance levels of the proposed methods are evaluated in experiments employing four real and two artificial datasets. Compared with the original learning vector quantization algorithms, our methods not only effectively remove the need for parameter tuning, but also achieve higher accuracy from learning small numbers of instances. In the cases of larger numbers of instances, SILVQ can still achieve an accuracy that is equal to or better than those of existing representative LVQ algorithms. Furthermore, SILVQ can learn linearly inseparable conceptual structures with the required and sufficient number of prototypes without overfitting.

Regenerative medicine: the challenge of building skeletal tissue A deeper understanding of skeletal tissue development and improvements in tissue engineering will help pluripotent stem cell (PSC) therapies to reach their full potential for skeletal repair. The paraxial mesoderm, an embryonic germ layer, is crucial to the formation of healthy axial skeleton. Shoichiro Tani at the University of Tokyo, Japan, and co-workers reviewed current understanding of paraxial mesoderm development and studies involving in vitro PSC skeletal modeling. The formation of the paraxial mesoderm and associated connective tissues comprises multiple stages, and studies in vertebrate embryos have uncovered critical signaling pathways and cellular components important to PSC modeling. Although many individual cellular components can now be modeled, it remains challenging to recreate three-dimensional skeletal tissues. Such an achievement would facilitate a functioning model of bone metabolism, the next step in achieving skeletal regeneration.

Clinically relevant human induced pluripotent stem cell (hiPSC) derivatives require efficient protocols to differentiate hiPSCs into specific lineages. Here we developed a fully defined xeno-free strategy to direct hiPSCs toward osteoblasts within 21 days. The strategy successfully achieved the osteogenic induction of four independently derived hiPSC lines by a sequential use of combinations of small-molecule inducers. The induction first generated mesodermal cells, which subsequently recapitulated the developmental expression pattern of major osteoblast genes and proteins. Importantly, Col2.3-Cherry hiPSCs subjected to this strategy strongly expressed the cherry fluorescence that has been observed in bone-forming osteoblasts in vivo. Moreover, the protocol combined with a three-dimensional (3D) scaffold was suitable for the generation of a xeno-free 3D osteogenic system. Thus, our strategy offers a platform with significant advantages for bone biology studies and it will also contribute to clinical applications of hiPSCs to skeletal regenerative medicine.

Abstract Purpose Chondrocyte -based tissue engineering has been a promising option for the treatment of cartilage lesions. In previous literature, TD198946 has been shown to promote chondrogenic differentiation which could prove useful in cartilage regeneration therapies. Our study aimed to investigate the effects of TD198946 in generating engineered cartilage using dedifferentiated chondrocyte-seeded collagen scaffolds treated with TD198946. Methods Articular chondrocytes were isolated from mini pig knees and expanded in 2-dimensional cell culture and subsequently used in the experiments. 3-D pellets were then cultured for two weeks. Cells were also cultured in a type I collagen scaffolds for four weeks. Specimens were cultured with TD198946, BMP-2, or both in combination. Outcomes were determined by gene expression levels of RUNX1, SOX9, ACAN, COL1A1, COL2A1 and COL10A1, the glycosaminoglycan content, and characteristics of histology and immunohistochemistry. Furthermore, the maturity of the engineered cartilage cultured for two weeks was evaluated through subcutaneous implantation in nude mice for four weeks. Results Addition of TD198946 demonstrated the upregulation of gene expression level except for ACAN, type II collagen and glycosaminoglycan synthesis in both pellet and 3D scaffold cultures. TD198946 and BMP-2 combination cultures showed higher chondrogenic differentiation than TD198946 or BMP-2 alone. The engineered cartilage maintained its extracellular matrices for four weeks post implantation. In contrast, engineered cartilage treated with either TD198946 or BMP-2 alone was mostly absorbed. Conclusions Our results indicate that TD198946 could improve quality of engineered cartilage by redifferentiation of dedifferentiated chondrocytes pre-implantation and promoting collagen and glycosaminoglycan synthesis.

BackgroundReducing the number of items in a questionnaire while maintaining relevant information is important as it is associated with advantages such as higher respondent engagement and reduced response error. However, in health care, after the original design, an a posteriori check of the included items in a questionnaire is often overlooked or considered to be of minor importance. When conducted, this is often based on a single selected method. We argue that before finalizing any lifestyle questionnaire, a posteriori validation should always be conducted using multiple approaches to ensure the robustness of the results. ObjectiveThe objectives of this study are to compare the results of two statistical methods for item reduction (variance inflation factor [VIF] and factor analysis [FA]) in a lifestyle questionnaire constructed by combining items from different sources and analyze the different results obtained from the 2 methods and the conclusions that can be made about the original items. MethodsData were collected from 79 participants (heterogeneous in age and sex) with a high risk of metabolic syndrome working in a financial company based in Tokyo. The lifestyle questionnaire was constructed by combining items (asked with daily, weekly, and monthly frequency) from multiple validated questionnaires and other selected questions. Item reduction was conducted using VIF and exploratory FA. Adequacy tests were used to check the data distribution and sampling adequacy. ResultsAmong the daily and weekly questions, both VIF and FA identified redundancies in sleep-related items. Among the monthly questions, both approaches identified redundancies in stress-related items. However, the number of items suggested for reduction often differed: VIF suggested larger reductions than FA for daily questions but fewer reductions for weekly questions. Adequacy tests always confirmed that the structural detection was adequate for the considered items. ConclusionsAs expected, our analyses showed that VIF and FA produced both similar and different findings, suggesting that questionnaire designers should consider using multiple methods for item reduction. Our findings using both methods indicate that many questions, especially those related to sleep, are redundant, indicating that the considered lifestyle questionnaire can be shortened.

The mobility of sustained molecules is influenced by viscoelasticity, which is strongly correlated with the diffusional property in polymeric liquid. However, the study of transient networks formed by a reversible crosslink, which is the viscoelastic liquid, was insufficient due to the absence of a model system. We compare the viscoelastic and diffusional properties of the transient networks, using the model system with controlled network connectivity (Tetra-PEG slime). According to independent measurements of viscoelasticity and diffusion, the root-mean-square distance the polymer diffuses during the viscoelastic relaxation time shows a large deviation from the self-size of the polymer, which is contrary to the conventional understanding. This decoupling between viscoelasticity and diffusion is unique for transient networks, suggesting that the viscoelastic relaxation is not induced by the diffusion of one prepolymer, particularly in the network with low connectivity. These findings will provide a definite basis for discussion to understand the viscoelasticity in transient networks.

For the application of polymer gels, it is necessary to control independently and precisely their various physical properties. However, the heterogeneity of polymer gels hinders the precise control over the structure, as well as the verification of theories. To understand the structure-property relationship of polymer gels, many researchers have tried to develop a homogeneous model network. Most of the model networks were made from polymer melts that did not have a solvent and had many entanglements in the structure. Because the contribution of entanglements is much larger than that of chemical crosslinking, it was difficult to focus on the crosslinking structure, which is the structure considered in conventional theories. To overcome such a situation, we have developed a new model network system that contains much solvent. Specifically, we fabricated the polymer gel (Tetra-PEG gel) by mixing two types of solutions of tetra-armed poly(ethylene glycol) (Tetra-PEG) with mutually reactive end groups (amine (-PA) and activated ester (-HS)). Because the existence of a solvent strongly reduces the effect of entanglements, the effect of the crosslinking structure on the physical properties can be extracted. In this review, we present the structure-property relationship of Tetra-PEG gel. First, we show the structural homogeneity of Tetra-PEG gels. Then, we explain gelation reaction, elastic modulus, fracture energy and kinetics of swelling and shrinking of Tetra-PEG gels by comparing the theories and experimental results.

Abstract Background Both loss- and gain-of-function of Wnt/β-catenin signaling in chondrocytes result in exacerbation of osteoarthritis (OA). Here, we examined the activity and roles of Wnt/β-catenin signaling in the superficial zone (SFZ) of articular cartilage. Methods Wnt/β-catenin signaling activity was analyzed using TOPGAL mice. We generated Prg4-Cre ERT2 ;Ctnnb1 fl/fl and Prg4-Cre ERT2 ;Ctnnb1-ex3 fl/wt mice for loss- and gain-of-function, respectively, of Wnt/β-catenin signaling in the SFZ. Regulation of Prg4 expression by Wnt/β-catenin signaling was examined in vitro, as were upstream and downstream factors of Wnt/β-catenin signaling in SFZ cells. Results Wnt/β-catenin signaling activity, as determined by the TOPGAL reporter, was high specifically in the SFZ of mouse adult articular cartilage, where Prg4 is abundantly expressed. In SFZ-specific β-catenin-knockout mice, OA development was significantly accelerated, which was accompanied by decreased Prg4 expression and SFZ destruction. In contrast, Prg4 expression was enhanced and cartilage degeneration was suppressed in SFZ-specific β-catenin-stabilized mice. In primary SFZ cells, Prg4 expression was downregulated by β-catenin knockout, while it was upregulated by β-catenin stabilization by exon 3 deletion or treatment with CHIR99021. Among Wnt ligands, Wnt5a, Wnt5b, and Wnt9a were highly expressed in SFZ cells, and recombinant human WNT5A and WNT5B stimulated Prg4 expression. Mechanical loading upregulated expression of these ligands and further promoted Prg4 transcription. Moreover, mechanical loading and Wnt/β-catenin signaling activation increased mRNA levels of Creb1, a potent transcription factor for Prg4. Conclusions We demonstrated that Wnt/β-catenin signaling regulates Prg4 expression in the SFZ of mouse adult articular cartilage, which plays essential roles in the homeostasis of articular cartilage.

Summary: A simple induction protocol to differentiate chondrocytes from pluripotent stem cells (PSCs) using small-molecule compounds is beneficial for cartilage regenerative medicine and mechanistic studies of chondrogenesis. Here, we demonstrate that chondrocytes are robustly induced from human PSCs by simple combination of two compounds, CHIR99021, a glycogen synthase kinase 3 inhibitor, and TTNPB, a retinoic acid receptor (RAR) agonist, under serum- and feeder-free conditions within 5–9 days. An excellent differentiation efficiency and potential to form hyaline cartilaginous tissues in vivo were demonstrated. Comprehensive gene expression and open chromatin analyses at each protocol stage revealed step-by-step differentiation toward chondrocytes. Genome-wide analysis of RAR and β-catenin association with DNA showed that retinoic acid and Wnt/β-catenin signaling collaboratively regulated the key marker genes at each differentiation stage. This method provides a promising cell source for regenerative medicine and, as an in vitro model, may facilitate elucidation of the molecular mechanisms underlying chondrocyte differentiation. : Saito and colleagues show that chondrocytes are robustly induced from human PSCs by simple combination of two compounds, a GSK3 inhibitor and an RAR agonist, within 5–9 days. Genome-wide analysis of RAR and β-catenin association with DNA for sequential samples at each protocol stage demonstrates that RA and Wnt/β-catenin signaling is collaboratively involved in direct regulation of chondrocyte differentiation. Keywords: pluripotent stem cells, cartilage, chondrocyte, chondrocyte differentiation, regenerative medicine, small-molecule compound

Excessive mechanical stress promotes the development of osteoarthritis. Here Chang et al. identify gremlin-1 as a factor expressed in chondrocytes in response to mechanical stress, and contributing to osteoarthritis via activation of the NF-κB pathway.

Identification of medical conditions using claims data is generally conducted with algorithms based on subject-matter knowledge. However, these claims-based algorithms (CBAs) are highly dependent on the knowledge level and not necessarily optimized for target conditions. We investigated whether machine learning methods can supplement researchers' knowledge of target conditions in building CBAs. Retrospective cohort study using a claims database combined with annual health check-up results of employees' health insurance programs for fiscal year 2016-17 in Japan (study population for hypertension, N = 631,289; diabetes, N = 152,368; dyslipidemia, N = 614,434). We constructed CBAs with logistic regression, k-nearest neighbor, support vector machine, penalized logistic regression, tree-based model, and neural network for identifying patients with three common chronic conditions: hypertension, diabetes, and dyslipidemia. We then compared their association measures using a completely hold-out test set (25% of the study population). Among the test cohorts of 157,822, 38,092, and 153,608 enrollees for hypertension, diabetes, and dyslipidemia, 25.4%, 8.4%, and 38.7% of them had a diagnosis of the corresponding condition. The areas under the receiver operating characteristic curve (AUCs) of the logistic regression with/without subject-matter knowledge about the target condition were .923/.921 for hypertension, .957/.938 for diabetes, and .739/.747 for dyslipidemia. The logistic lasso, logistic elastic-net, and tree-based methods yielded AUCs comparable to those of the logistic regression with subject-matter knowledge: .923-.931 for hypertension; .958-.966 for diabetes; .747-.773 for dyslipidemia. We found that machine learning methods can attain AUCs comparable to the conventional knowledge-based method in building CBAs.

Book Review of “The Righteous Mind-Why Good People are Divided by Politics and Religion” Written by Jonathan Haidt (2012). New York: Pantheon Books. eISBN: 978-0-307-90703-5.

Bayesian inference is the process of narrowing down the hypotheses (causes) to the one that best explains the observational data (effects). To accurately estimate a cause, a considerable amount of data is required to be observed for as long as possible. However, the object of inference is not always constant. In this case, a method such as exponential moving average (EMA) with a discounting rate is used to improve the ability to respond to a sudden change; it is also necessary to increase the discounting rate. That is, a trade-off is established in which the followability is improved by increasing the discounting rate, but the accuracy is reduced. Here, we propose an extended Bayesian inference (EBI), wherein human-like causal inference is incorporated. We show that both the learning and forgetting effects are introduced into Bayesian inference by incorporating the causal inference. We evaluate the estimation performance of the EBI through the learning task of a dynamically changing Gaussian mixture model. In the evaluation, the EBI performance is compared with those of the EMA and a sequential discounting expectation-maximization algorithm. The EBI was shown to modify the trade-off observed in the EMA.

The 17th Congress of the Japanese Society for Regenerative Medicine was held on March 21–23, 2018 at PACIFICO Yokohama (Kanagawa Prefecture) with 3860 participants. The theme of the congress was ‘The Integration of Wisdom from All Sectors.’ With this theme, this congress aimed to provide people from all sectors (including individuals from various industries, regulatory authorities, academia, and citizens) with opportunities for exchanging views on regenerative medicine under one roof. A broad spectrum of topics related to regenerative medicine was covered by one presidential lecture, one keynote lecture, one collaborative lecture by the Congress Chair and the Governor of Kanagawa Prefecture, three special lectures (six topics), four award lectures, 43 symposia (235 talks), 337 oral presentations (59 sessions), 358 poster presentations (43 sessions), 25 co-organized seminars (35 talks), two sessions for junior high school and high school students (basic and advanced), and the state-of-the-art technology showcase (158 organizations). Keywords: Congress report, Regenerative medicine

Abstract Background Human dental pulp stem cells (DPSCs), which have the ability to differentiate into multiple lineages, were recently identified. DPSCs can be collected readily from extracted teeth and are now considered to be a type of mesenchymal stem cell with higher clonogenic and proliferative potential than bone marrow stem cells (BMSCs). Meanwhile, the treatment of severe bone defects, such as fractures, cancers, and congenital abnormalities, remains a great challenge, and novel bone regenerative techniques are highly anticipated. Several studies have previously shown that 4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2,3-b]pyridine-2-carboxamide (TH), a helioxanthin derivative, induces osteogenic differentiation of preosteoblastic and mesenchymal cells. However, the osteogenic differentiation activities of TH have only been confirmed in some mouse cell lines. Therefore, in this study, toward the clinical use of TH in humans, we analyzed the effect of TH on the osteogenic differentiation of DPSCs, and the in-vivo osteogenesis ability of TH-induced DPSCs, taking advantage of the simple transplantation system using cell-sheet technology. Methods DPSCs were obtained from dental pulp of the wisdom teeth of five healthy patients (18–22 years old) and cultured in regular medium and osteogenic medium with or without TH. To evaluate osteogenesis of TH-induced DPSCs in vivo, we transplanted DPSC sheets into mouse calvaria defects. Results We demonstrated that osteogenic conditions with TH induce the osteogenic differentiation of DPSCs more efficiently than those without TH and those with bone morphogenetic protein-2. However, regular medium with TH did not induce the osteogenic differentiation of DPSCs. TH induced osteogenesis in both DPSCs and BMSCs, although the gene expression pattern in DPSCs differed from that in BMSCs up to 14 days after induction with TH. Furthermore, we succeeded in bone regeneration in vivo using DPSC sheets with TH treatment, without using any scaffolds or growth factors. Conclusions Our results demonstrate that TH-induced DPSCs are a useful cell source for bone regenerative medicine, and the transplantation of DPSC sheets treated with TH is a convenient scaffold-free method of bone healing.

Communication between osteoblasts and osteoclasts is essential for bone homeostasis, but the mode of interaction is unclear. The authors use intravital two-photon microscopy in mice to show that these cells directly interact, regulating activity of osteoclasts, and that the interaction is modulated by parathyroid hormone administration.

Physiological time series are affected by many factors, making them highly nonlinear and nonstationary. As a consequence, heart rate time series are often considered difficult to predict and handle. However, heart rate behavior can indicate underlying cardiovascular and respiratory diseases as well as mood disorders. Given the importance of accurate modeling and reliable predictions of heart rate fluctuations for the prevention and control of certain diseases, it is paramount to identify models with the best performance in such tasks. The objectives of this study were to compare the results of three different forecasting models (Autoregressive Model, Long Short-Term Memory Network, and Convolutional Long Short-Term Memory Network) trained and tested on heart rate beats per minute data obtained from twelve heterogeneous participants and to identify the architecture with the best performance in terms of modeling and forecasting heart rate behavior. Heart rate beats per minute data were collected using a wearable device over a period of 10 days from twelve different participants who were heterogeneous in age, sex, medical history, and lifestyle behaviors. The goodness of the results produced by the models was measured using both the mean absolute error and the root mean square error as error metrics. Despite the three models showing similar performance, the Autoregressive Model gave the best results in all settings examined. For example, considering one of the participants, the Autoregressive Model gave a mean absolute error of 2.069 (compared to 2.173 of the Long Short-Term Memory Network and 2.138 of the Convolutional Long Short-Term Memory Network), achieving an improvement of 5.027% and 3.335%, respectively. Similar results can be observed for the other participants. The findings of the study suggest that regardless of an individual’s age, sex, and lifestyle behaviors, their heart rate largely depends on the pattern observed in the previous few minutes, suggesting that heart rate can be reasonably regarded as an autoregressive process. The findings also suggest that minute-by-minute heart rate prediction can be accurately performed using a linear model, at least in individuals without pathologies that cause heartbeat irregularities. The findings also suggest many possible applications for the Autoregressive Model, in principle in any context where minute-by-minute heart rate prediction is required (arrhythmia detection and analysis of the response to training, among others).

A class of gene-regulatory elements called enhancers are the main mediators controlling quantitative, temporal and spatial gene expressions. In the course of evolution, the enhancer landscape of higher organisms such as mammals has become quite complex, exerting biological functions precisely and coordinately. In mammalian skeletal development, the master transcription factors Sox9, Runx2 and Sp7/Osterix function primarily through enhancers on the genome to achieve specification and differentiation of skeletal cells. Recently developed genome-scale analyses have shed light on multiple layers of gene regulations, uncovering not only the primary mode of actions of these transcription factors on skeletal enhancers, but also the relation of the epigenetic landscape to three-dimensional chromatin architecture. Here, we review findings on the emerging framework of gene-regulatory networks involved in skeletal development. We further discuss the power of genome-scale analyses to provide new insights into genetic diseases and regenerative medicine in skeletal tissues.

The frequency of occurrence of step length in the migratory behaviour of various organisms, including humans, is characterized by the power law distribution. This pattern of behaviour is known as the Lévy walk, and the reason for this phenomenon has been investigated extensively. Especially in humans, one possibility might be that this pattern reflects the change in self-confidence in one’s chosen behaviour. We used simulations to demonstrate that active assumptions cause changes in the confidence level in one’s choice under a situation of lack of information. More specifically, we presented an algorithm that introduced the effects of learning and forgetting into Bayesian inference, and simulated an imitation game in which two decision-making agents incorporating the algorithm estimated each other’s internal models. For forgetting without learning, each agents’ confidence levels in their own estimation remained low owing to a lack of information about the counterpart, and the agents changed their hypotheses about the opponent frequently, and the frequency distribution of the duration of the hypotheses followed an exponential distribution for a wide range of forgetting rates. Conversely, when learning was introduced, high confidence levels occasionally occurred even at high forgetting rates, and exponential distributions universally turned into power law distribution.

Polyelectrolyte gels exhibit swelling behaviors that are dependent on the external environment. The swelling behaviors of highly charged polyelectrolyte gels can be well explained using the Flory–Rehner model combined with the Gibbs–Donnan effect and Manning’s counterion condensation effect (the FRGDM model). This study investigated the swelling properties of a series of model polyelectrolyte gels, namely tetra-polyacrylic acid-polyethylene glycol gels (Tetra-PAA-PEG gels), and determined the applicability of the FRGDM model. The swelling ratio (Vs/V0) was well reproduced by the FRGDM model in the moderate swelling regime (Vs/V0 < 10). However, in the high swelling regime (Vs/V0 > 10), the FRGDM model is approx. 1.6 times larger than the experimental results. When we introduced the finite extensibility to the elastic free energy in the FRGDM model, the swelling behavior was successfully reproduced even in the high swelling regime. Our results reveal that finite extensibility is one of the factors determining the swelling equilibrium of highly charged polyelectrolyte gels. The modified FRGDM model reproduces well the swelling behavior of a wide range of polyelectrolyte gels.

Rubber elasticity is the archetype of the entropic force emerging from the second law of thermodynamics; numerous experimental and theoretical studies on natural and synthetic rubbers have shown that the elasticity originates mostly from entropy change with deformation. Similarly, in polymer gels containing a large amount of solvent, it has also been postulated that the shear modulus (the modulus of rigidity) G, which is a kind of modulus of elasticity, is approximately equivalent to the entropy contribution G_{S}, but this has yet to be verified experimentally. In this study, we measure the temperature dependence of the shear modulus G in a rubberlike (hyperelastic) polymer gel whose polymer volume fraction is at most 0.1. As a result, we find that the energy contribution G_{E}=G-G_{S} can be a significant negative value, reaching up to double the shear modulus G (i.e., |G_{E}|≃2G), although the shear modulus of stable materials is generally bound to be positive. We further argue that the energy contribution G_{E} is governed by a vanishing temperature that is a universal function of the normalized polymer concentration, and G_{E} vanishes when the solvent is removed. Our findings highlight the essential difference between rubber elasticity and gel elasticity (which were previously thought to be the same) and push the established field of gel elasticity into a new direction.

Rela is a transcription factor shown to have seemingly contradictory roles in anabolism and catabolism of cartilage. Here the authors find that Rela prevents chondrocyte apoptosis and that homozygous knockout causes accelerated osteoarthritis in adults, whereas heterozygous knockout suppresses osteoarthritis by maintaining wild-type effects on apoptosis but inhibiting catabolic gene expression.

Tissue engineering is an approach to the regeneration of tissues that uses a combination of cell sources, signaling factors and scaffolds. Among these three components, signaling factors for bone regeneration have not yet been established, and it is necessary to better understand osteoblast progenitors as a target cells. Several lines of evidence have revealed that, during bone formation, mesenchymal cells are specified and differentiate into osteoblasts through several stages of precursors. The osteoblast lineage is defined by the expression of stage-specific transcription factors. The specification and differentiation are organized by a variety of signaling pathways including hedgehog (Hh), Wnt, Notch, bone morphogenetic protein (BMP) and transforming growth factor-beta (TGFβ). In this review we integrate the known functions of these signaling pathways and discuss future tasks to gain a better understanding of the signaling network in osteogenesis for tissue engineering.

Although techniques for cell-specific gene expression via viral transfer have advanced, many challenges (e.g., viral vector design, transduction of genes into specific target cells) still remain. We investigated a novel, simple methodology for using adenovirus transfer to target specific cells of the kidney tubules for the expression of exogenous proteins. We selected genes encoding sodium-dependent phosphate transporter type 2a (NPT2a) in the proximal tubule, sodium-potassium-2-chloride cotransporter (NKCC2) in the thick ascending limb of Henle (TALH), and aquaporin 2 (AQP2) in the collecting duct. The promoters of the three genes were linked to a GFP-coding fragment, the final constructs were then incorporated into an adenovirus vector, and this was then used to generate gene-manipulated viruses. After flushing circulating blood, viruses were directly injected into the renal arteries of rats and were allowed to site-specifically expression in tubule cells, and rats were then euthanized to obtain kidney tissues for immunohistochemistry. Double staining with adenovirus-derived EGFP and endogenous proteins were examined to verify orthotopic expression, i.e. "adenovirus driven NPT2a-EGFP and endogenous NHE3 protein", "adenovirus driven NKCC2-EGFP and endogenous NKCC2 protein" and "adenovirus driven AQP2-EGFP and endogenous AQP2 protein". Owing to a lack of finding good working anti-NPT2a antibody, an antibody against a different protein (sodium-hydrogen exchanger 3 or NHE3) that is also specifically expressed in the proximal tubule was used. Kidney structures were well-preserved, and other organ tissues did not show EGFP staining. Our gene transfer method is easier than using genetically engineered animals, and it confers the advantage of allowing the manipulation of gene transfer after birth. This is the first method to successfully target gene expression to specific cells in the kidney tubules. This study may serve as the first step for safe and effective gene therapy in the kidney tubule diseases.

The apical ectodermal ridge (AER), located at the distal end of each limb bud, is a key signaling center which controls outgrowth and patterning of the proximal-distal axis of the limb through secretion of various molecules. Fibroblast growth factors (FGFs), particularly Fgf8 and Fgf4, are representative molecules produced by AER cells, and essential to maintain the AER and cell proliferation in the underlying mesenchyme, meanwhile Jag2-Notch pathway negatively regulates the AER and limb development. p63, a transcription factor of the p53 family, is expressed in the AER and indispensable for limb formation. However, the underlying mechanisms and specific roles of p63 variants are unknown. Here, we quantified the expression of p63 variants in mouse limbs from embryonic day (E) 10.5 to E12.5, and found that ΔNp63γ was strongly expressed in limbs at all stages, while TAp63γ expression was rapidly increased in the later stages. Fluorescence-activated cell sorting analysis of limb bud cells from reporter mouse embryos at E11.5 revealed that all variants were abundantly expressed in AER cells, and their expression was very low in mesenchymal cells. We then generated AER-specific p63 knockout mice by mating mice with a null and a flox allele of p63, and Msx2-Cre mice (Msx2-Cre;p63Δ/fl). Msx2-Cre;p63Δ/fl neonates showed limb malformation that was more obvious in distal elements. Expression of various AER-related genes was decreased in Msx2-Cre;p63Δ/fl limb buds and embryoid bodies formed by p63-knockdown induced pluripotent stem cells. Promoter analyses and chromatin immunoprecipitation assays demonstrated Fgf8 and Fgf4 as transcriptional targets of ΔNp63γ, and Jag2 as that of TAp63γ. Furthermore, TAp63γ overexpression exacerbated the phenotype of Msx2-Cre;p63Δ/fl mice. These data indicate that ΔNp63 and TAp63 control limb development through transcriptional regulation of different target molecules with different roles in the AER. Our findings contribute to further understanding of the molecular network of limb development.

Pluripotent stem cells are a promising tool for mechanistic studies of tissue development, drug screening, and cell-based therapies. Here, we report an effective and mass-producing strategy for the stepwise differentiation of mouse embryonic stem cells (mESCs) and mouse and human induced pluripotent stem cells (miPSCs and hiPSCs, respectively) into osteoblasts using four small molecules (CHIR99021 [CHIR], cyclopamine [Cyc], smoothened agonist [SAG], and a helioxanthin-derivative 4-(4-methoxyphenyl)pyrido[4′,3′:4,5]thieno[2,3-b]pyridine-2-carboxamide [TH]) under serum-free and feeder-free conditions. The strategy, which consists of mesoderm induction, osteoblast induction, and osteoblast maturation phases, significantly induced expressions of osteoblast-related genes and proteins in mESCs, miPSCs, and hiPSCs. In addition, when mESCs defective in runt-related transcription factor 2 (Runx2), a master regulator of osteogenesis, were cultured by the strategy, they molecularly recapitulated osteoblast phenotypes of Runx2 null mice. The present strategy will be a platform for biological and pathological studies of osteoblast development, screening of bone-augmentation drugs, and skeletal regeneration.

Present titanium plates for implants have the problems like too heavy and excessive elastic modulus compared with natural bones. In this study, Titanium mesh plates with higher 3-demensional flexibility were developed to improve such problems. Fundamental mesh patterns were designed through three dimensional CAD tools from higher flexibility and strength points of view. Based on the designed mesh patterns, sample specimens of Titanium mesh plates with different design variables like plate thickness, mesh line width were manufactured through wire-cut discharge processing. Mechanical properties of the Titanium mesh plates like volume density, tensile elastic modulus and bending stiffness were experimentally and analytically evaluated. Experimental results showed that such Titanium mesh plates had much higher flexibility and their mechanical properties could be controlled to close to the natural bones. More details on the mechanical properties of meshed plates including compression, torsion and durability will be executed in future study.

Transcription factors SOX9, SOX5 and SOX6 are indispensable for generation and differentiation of chondrocytes. However, molecular mechanisms to induce the SOX genes are poorly understood. To address this issue, we previously determined the human embryonic enhancer of SOX6 by 5'RACE analysis, and identified the 46-bp core enhancer region (CES6). We initially performed yeast one-hybrid assay for screening other chondrogenic factors using CES6 as bait, and identified a zinc finger protein ZNF449. ZNF449 and Zfp449, a counterpart in mouse, transactivated enhancers or promoters of SOX6, SOX9 and COL2A1. Zfp449 was expressed in mesenchyme-derived tissues including cartilage, calvaria, muscle and tendon, as well as in other tissues including brain, lung and kidney. In limb cartilage of mouse embryo, Zfp449 protein was abundantly located in periarticular chondrocytes, and decreased in accordance with the differentiation. Zfp449 protein was also detected in articular cartilage of an adult mouse. During chondrogenic differentiation of human mesenchymal stem cells, ZNF449 was increased at an early stage, and its overexpression enhanced SOX9 and SOX6 only at the initial stage of the differentiation. We further generated Zfp449 knockout mice to examine the in vivo roles; however, no obvious abnormality was observed in skeletal development or articular cartilage homeostasis. ZNF449 may regulate chondrogenic differentiation from mesenchymal progenitor cells, although the underlying mechanisms are still unknown.

Hedgehog (Hh) signaling plays important roles in various development processes. This signaling is necessary for osteoblast formation during endochondral ossification. In contrast to the established roles of Hh signaling in embryonic bone formation, evidence of its roles in adult bone homeostasis is not complete. Here we report the involvement of Gli1, a transcriptional activator induced by Hh signaling activation, in postnatal bone homeostasis under physiological and pathological conditions. Skeletal analyses of Gli1+/- adult mice revealed that Gli1 haploinsufficiency caused decreased bone mass with reduced bone formation and accelerated bone resorption, suggesting an uncoupling of bone metabolism. Hh-mediated osteoblast differentiation was largely impaired in cultures of Gli1+/- precursors, and the impairment was rescued by Gli1 expression via adenoviral transduction. In addition, Gli1+/- precursors showed premature differentiation into osteocytes and increased ability to support osteoclastogenesis. When we compared fracture healing between wild-type and Gli1+/- adult mice, we found that the Gli1+/- mice exhibited impaired fracture healing with insufficient soft callus formation. These data suggest that Gli1, acting downstream of Hh signaling, contributes to adult bone metabolism, in which this molecule not only promotes osteoblast differentiation but also represses osteoblast maturation toward osteocytes to maintain normal bone homeostasis.

Tenomodulin (Tnmd) is a type II transmembrane protein characteristically expressed in dense connective tissues such as tendons and ligaments. Its expression in the periodontal ligament (PDL) has also been demonstrated, though the timing and function remain unclear. We investigated the expression of Tnmd during murine tooth eruption and explored its biological functions in vitro. Tnmd expression was related to the time of eruption when occlusal force was transferred to the teeth and surrounding tissues. Tnmd overexpression enhanced cell adhesion in NIH3T3 and human PDL cells. In addition, Tnmd-knockout fibroblasts showed decreased cell adhesion. In the extracellular portions of Tnmd, the BRICHOS domain or CS region was found to be responsible for Tnmd-mediated enhancement of cell adhesion. These results suggest that Tnmd acts on the maturation or maintenance of the PDL by positively regulating cell adhesion via its BRICHOS domain.

Induced pluripotent stem cells (iPSC) are a promising cell source for cartilage regenerative medicine; however, the methods for chondrocyte induction from iPSC are currently developing and not yet sufficient for clinical application. Here, we report the establishment of a fluorescent indicator system for monitoring chondrogenic differentiation from iPSC to simplify screening for effective factors that induce chondrocytes from iPSC. We generated iPSC from embryonic fibroblasts of Col2a1-EGFP transgenic mice by retroviral transduction of Oct4, Sox2, Klf4, and c-Myc. Among the 30 clones of Col2a1-EGFP iPSC we established, two clones showed high expression levels of embryonic stem cell (ESC) marker genes, similar to control ESC. A teratoma formation assay showed that the two clones were pluripotent and differentiated into cell types from all three germ layers. The fluorescent signal was observed during chondrogenic differentiation of the two clones concomitant with the increase in chondrocyte marker expression. In conclusion, Col2a1-EGFP iPSC are useful for monitoring chondrogenic differentiation and will contribute to research in cartilage regenerative medicine.

BACKGROUND: Although transition from proliferation to hypertrophic differentiation of chondrocytes is a crucial step for endochondral ossification in physiological skeletal growth and pathological disorders like osteoarthritis, the underlying mechanism remains an enigma. This study investigated the role of the transcription factor CCAAT/enhancer-binding protein beta (C/EBPbeta) in chondrocytes during endochondral ossification. METHODOLOGY/PRINCIPAL FINDINGS: Mouse embryos with homozygous deficiency in C/EBPbeta (C/EBPbeta-/-) exhibited dwarfism with elongated proliferative zone and delayed chondrocyte hypertrophy in the growth plate cartilage. In the cultures of primary C/EBPbeta-/- chondrocytes, cell proliferation was enhanced while hypertrophic differentiation was suppressed. Contrarily, retroviral overexpression of C/EBPbeta in chondrocytes suppressed the proliferation and enhanced the hypertrophy, suggesting the cell cycle arrest by C/EBPbeta. In fact, a DNA cell cycle histogram revealed that the C/EBPbeta overexpression caused accumulation of cells in the G0/G1 fraction. Among cell cycle factors, microarray and real-time RT-PCR analyses have identified the cyclin-dependent kinase inhibitor p57(Kip2) as the transcriptional target of C/EBPbeta. p57(Kip2) was co-localized with C/EBPbeta in late proliferative and pre-hypertrophic chondrocytes of the mouse growth plate, which was decreased by the C/EBPbeta deficiency. Luciferase-reporter and electrophoretic mobility shift assays identified the core responsive element of C/EBPbeta in the p57(Kip2) promoter between -150 and -130 bp region containing a putative C/EBP motif. The knockdown of p57(Kip2) by the siRNA inhibited the C/EBPbeta-induced chondrocyte hypertrophy. Finally, when we created the experimental osteoarthritis model by inducing instability in the knee joints of adult mice of wild-type and C/EBPbeta+/- littermates, the C/EBPbeta insufficiency caused resistance to joint cartilage destruction. CONCLUSIONS/SIGNIFICANCE: C/EBPbeta transactivates p57(Kip2) to promote transition from proliferation to hypertrophic differentiation of chondrocytes during endochondral ossification, suggesting that the C/EBPbeta-p57(Kip2) signal would be a therapeutic target of skeletal disorders like growth retardation and osteoarthritis.

Bone mass and turnover are maintained by the coordinated balance between bone formation by osteoblasts and bone resorption by osteoclasts, under regulation of many systemic and local factors. Phosphoinositide-dependent serine-threonine protein kinase Akt is one of the key players in the signaling of potent bone anabolic factors. This study initially showed that the disruption of Akt1, a major Akt in osteoblasts and osteoclasts, in mice led to low-turnover osteopenia through dysfunctions of both cells. Ex vivo cell culture analyses revealed that the osteoblast dysfunction was traced to the increased susceptibility to the mitochondria-dependent apoptosis and the decreased transcriptional activity of runt-related transcription factor 2 (Runx2), a master regulator of osteoblast differentiation. Notably, our findings revealed a novel role of Akt1/forkhead box class O (FoxO) 3a/Bim axis in the apoptosis of osteoblasts: Akt1 phosphorylates the transcription factor FoxO3a to prevent its nuclear localization, leading to impaired transactivation of its target gene Bim which was also shown to be a potent proapoptotic molecule in osteoblasts. The osteoclast dysfunction was attributed to the cell autonomous defects of differentiation and survival in osteoclasts and the decreased expression of receptor activator of nuclear factor-kappaB ligand (RANKL), a major determinant of osteoclastogenesis, in osteoblasts. Akt1 was established as a crucial regulator of osteoblasts and osteoclasts by promoting their differentiation and survival to maintain bone mass and turnover. The molecular network found in this study will provide a basis for rational therapeutic targets for bone disorders.

Despite accumulated knowledge of various signalings regulating bone formation, the molecular network has not been clarified sufficiently to lead to clinical application. Here we show that heterozygous glycogen synthase kinase-3beta (GSK-3beta)-deficient mice displayed an increased bone formation due to an enhanced transcriptional activity of Runx2 by suppressing the inhibitory phosphorylation at a specific site. The cleidocranial dysplasia in heterozygous Runx2-deficient mice was significantly rescued by the genetic insufficiency of GSK-3beta or the oral administration of lithium chloride, a selective inhibitor of GSK-3beta. These results establish GSK-3beta as a key attenuator of Runx2 activity in bone formation and as a potential molecular target for clinical treatment of bone catabolic disorders like cleidocranial dysplasia.