Published papers

Severe dental tissue damage induces odontoblast death, after which dental pulp stem and progenitor cells (DPSCs) differentiate into odontoblast-like cells, contributing to reparative dentin. However, the damage-induced mechanism that triggers this regeneration process is still not clear. We aimed to understand the effect of odontoblast death without hard tissue damage on dental regeneration. Herein, using a Cre/LoxP-based strategy, we demonstrated that cell-rich zone (CZ)-localizing Nestin-GFP-positive and Nestin-GFP-negative cells proliferate and differentiate into odontoblast-like cells in response to odontoblast depletion. The regenerated odontoblast-like cells played a role in reparative dentin formation. RNA-sequencing analysis revealed that the expression of odontoblast differentiation- and activation-related genes was upregulated in the pulp in response to odontoblast depletion even without damage to dental tissue. In this regenerative process, the expression of type I parathyroid hormone receptor (PTH1R) increased in the odontoblast-depleted pulp, thereby boosting dentin formation. The levels of PTH1R and its downstream mediator, i.e., phosphorylated cyclic AMP response element-binding protein (Ser133) increased in the physically damaged pulp. Collectively, odontoblast death triggered the PTH1R cascade, which may represent a therapeutic target for inducing CZ-mediated dental regeneration.

The locomotor system is highly bilateral at the macroscopic level. Homochirality of biological molecules is fully compatible with the bilateral body. However, whether and how single-handed cells contribute to the bilateral locomotor system is obscure. Here, exploiting the small number of cells in the swimming tadpole larva of the ascidian Ciona, we analyzed morphology of the tail at cellular and subcellular scales. Quantitative phase-contrast X-ray tomographic microscopy revealed a high-density midline structure ventral to the notochord in the tail. Muscle cell nuclei on each side of the notochord were roughly bilaterally aligned. However, fluorescence microscopy detected left-right asymmetry of myofibril inclination relative to the longitudinal axis of the tail. Zernike phase-contrast X-ray tomographic microscopy revealed the presence of left-handed helices of myofibrils in muscle cells on both sides. Therefore, the locomotor system of ascidian larvae harbors symmetry-breaking left-handed helical cells, while maintaining bilaterally symmetrical cell alignment. These results suggest that bilateral animals can override cellular homochirality to generate the bilateral locomotor systems at the supracellular scale.

Vertebrates are bilateral organisms characterized by both symmetrical and asymmetrical structures. While skeletons are grossly symmetrical, microscopic biological networks within the bone—such as cortical canals containing vascular networks—often exhibit deviations from precise mirror-image symmetry. These deviations make it challenging to detect subtle chiral biases amidst random structural variations. We developed the “bipixel rotation method” to quantify the extent of these biases, thereby enabling the characterization of porcine femoral cortical canals. In this approach, binarized 2D images are rotated stepwise and skeletonized, followed by the quantification of vertically adjacent pixel pairs. We defined “center angle” as the rotation angle that yields the peak vertical bipixel count, and calculated “down-right bias” based on the asymmetry of the distribution of vertical bipixels around this center angle. After validating the method using alphabetical fonts and geometric wallpaper patterns, we applied the method to the cortical canal layers in porcine fibrolamellar bone visualized via nano-computed tomography (nano-CT). The method successfully detected potential chirality in the cortical canals, identifying up-right versus down-right biases, demonstrating its capability to capture even subtle structural chirality. These results suggest that the bipixel method is a versatile tool for chirality analysis of diverse biological mesh structures in the body.

Calcified cartilage digested by chondroclasts provides an excellent scaffold to initiate bone formation. We analyzed bioactive proteins and microarchitecture of calcified cartilage either separately or in combination and evaluated biomimetic osteogenic culture conditions of surface-coated micropatterning. To do so, we prepared a crude extract from porcine femoral growth plates, which enhanced in vitro mineralization when coated on flat-bottom culture dishes, and identified four candidate proteins by fractionation and mass spectrometry. Murine homologues of two candidates, desmoglein 4 (DSG4) and peroxiredoxin 6 (PRDX6), significantly promoted osteogenic activity based on in vitro mineralization and osteoblast differentiation. Moreover, we observed DSG4 and PRDX6 protein expression in mouse femur. In addition, we designed circular, triangular, and honeycomb micropatterns with 30 or 50 μm units, either isolated or connected, to mimic hypertrophic chondrocyte-sized compartments. Isolated, larger honeycomb patterns particularly enhanced osteogenesis in vitro. Mineralization on micropatterns was positively correlated with the reduction of osteoblast migration distance in live cell imaging. Finally, we evaluated possible combinatorial effects of coat proteins and micropatterns and observed an additive effect of DSG4 or PRDX6 coating with micropatterns. These data suggest that combining a bioactive surface coating with osteogenic micropatterns may recapitulate initiation of bone formation during endochondral ossification.

Purpose: Chordomas are malignant tumors that destroy bones, compress surrounding nerve tissues and exhibit phenotypes that recapitulate notochordal differentiation in the axial skeleton. Chordomas recur frequently, as they resist radio-chemotherapy and are difficult to completely resect, leading to repeated bone destruction and local expansion via unknown mechanisms. Here, using chordoma specimens and JHC7 chordoma cells, we asked whether chordoma cells possess bone-dissolving activity. Methods: CT imaging and histological analysis were performed to evaluate the structure and mineral density of chordoma-invaded bone and osteolytic marker expression. JHC7 cells were subjected to immunocytochemistry, imaging of cell fusion, calcium dynamics and acidic vacuoles, and bone lysis assays. Results: In patients, we found that the skull base invaded by chordoma was highly porous, showed low mineral density and contained brachyury-positive chordoma cells and conventional osteoclasts both expressing the osteolytic markers tartrate-resistant acid phosphatase (TRAP) and collagenases. JHC7 cells expressed TRAP and cathepsin K, became multinucleated via cell-cell fusion, showed spontaneous calcium oscillation, and were partly responsive to the osteoclastogenic cytokine RANKL. JHC7 cells exhibited large acidic vacuoles, and nonregulatory bone degradation without forming actin rings. Finally, bone-derived factors, calcium ions, TGF-β1, and IGF-1 enhanced JHC7 cell proliferation. Conclusion: In chordoma, we propose that in addition to conventional bone resorption by osteoclasts, chordoma cells possess bone-dissolving activity at the tumor-bone boundary. Furthermore, bone destruction and tumor expansion may occur in a positive feedback loop.

Hereditary hearing loss is highly heterogeneous. SLC12A2 is linked to autosomal dominant nonsyndromic hearing loss, DFNA78, with all the pathogenic variants affecting the exon 21. The gene encodes a cotransporter NKCC1 crucial for regulating intracellular osmotic pressure and producing endolymph in the cochlea. We generated two mouse strains with heterologous Slc12a2 variants in the splice site of the exon 21 (Em1: NM_009194.3:c.2912-2 A > G and Em2: c.2912-4_2913del). Slc12a2Em2/Em2 mice with complete skip of the exon 21 showed reduced endolymph on postnatal day 1 (P1), reduced stria vascularis (StV) and no auditory brainstem responses at 4 weeks. Reduced StV size was considered to be due to rebalance osmotic pressure, and upregulation of Cldn9 revealed by RNA-seq was considered as tissue response to repair the gaps from reduced cell sizes in the Slc12a2Em2/Em2 cochlea. Female Slc12a2Em2/+ mice also exhibited mild elevation of ABR thresholds in several sound frequencies. Slc12a2Em1/Em1 mice showed normal hearing, presumably due to sufficient cotransporter activity from the 9 bases shorter transcript by cryptic splicing. Minigene assays indicated that a single nucleotide difference between humans and mice at the 5’ end of the exon 21 affects exon 21 splicing. Slc12a2Em2 mouse is proposed as a model for studying DFNA78 pathology.

Developing long bones alter their shape while maintaining uniform cortical thickness via coordinated activity of bone-forming osteoblasts and bone-resorbing osteoclasts at periosteal and endosteal surfaces, a process we designate trans-pairing. Two types of trans-pairing shift cortical bone in opposite orientations: peri-forming trans-pairing (peri-t-p) increases bone marrow space and endo-forming trans-pairing (endo-t-p) decreases it, via paired activity of bone resorption and formation across the cortex. Here, we focused on endo-t-p in growing bones. Analysis of endo-t-p activity in the cortex of mouse fibulae revealed osteoclasts under the periosteum compressed by muscles, and expression of RANKL in periosteal cells of the cambium layer. Furthermore, mature osteoblasts were localized on the endosteum, while preosteoblasts were at the periosteum and within cortical canals. X-ray tomographic microscopy revealed the presence of cortical canals more closely associated with endo- than with peri-t-p. Sciatic nerve transection followed by muscle atrophy and unloading induced circumferential endo-t-p with concomitant spread of cortical canals. Such canals likely supply the endosteum with preosteoblasts from the periosteum under endo-t-p, allowing bone shape to change in response to mechanical stress or nerve injury.

Introduction: The conditional manipulation of genes using the Cre recombinase-locus of crossover in P1 (Cre/loxP) system is an important tool for revealing gene functions and cell lineages in vivo. The outcome of this method is dependent on the performance of Cre-driver mouse strains. In most cases, Cre knock-in mice show better specificity than randomly inserted Cre transgenic mice. However, following knock-in, the expression of the original gene replaced by Cre is lost. Materials and methods: We generated a new differentiated osteoblast- and osteocyte-specific Cre knock-in mouse line that carries the viral T2A sequence encoding a 2A self-cleaving peptide at the end of the coding region of the dentin matrix protein 1 (Dmp1) gene accompanied by the Cre gene. Results: We confirmed that Dmp1-T2A-Cre mice showed high Cre expression in osteoblasts, osteocytes, odontoblasts, and periodontal ligament cells and that the 2A self-cleaving peptide efficiently produced both Dmp1 and Cre proteins. Furthermore, unlike the Dmp1 knockout mice, homozygous Dmp1-T2A-Cre mice showed no skeletal abnormalities. Analysis using the Cre reporter strain confirmed differentiated osteoblast- and osteocyte-specific Cre-mediated recombination in the skeleton. Furthermore, recombination was also detected in some nuclei of skeletal muscle cells, spermatocytes, and intestinal cells. Conclusion: 2A-Cre functions effectively in vivo, and Dmp1-T2A-Cre knock-in mice are a useful tool for studying the functioning of various genes in hard tissues.

In this study, high-energy x-ray nanotomography (nano-computed tomography, nano-CT) based on full-field x-ray microscopy was developed. Fine two-dimensional and three-dimensional (3D) structures with linewidths of 75 nm-100 nm were successfully resolved in the x-ray energy range of 15 keV-37.7 keV. The effective field of view was ∼60 μm, and the typical measurement time for one tomographic scan was 30 min-60 min. The optical system was established at the 250-m-long beamline 20XU of SPring-8 to realize greater than 100× magnification images. An apodization Fresnel zone plate (A-FZP), specifically developed for high-energy x-ray imaging, was used as the objective lens. The design of the A-FZP for high-energy imaging is discussed, and its diffraction efficiency distribution is evaluated. The spatial resolutions of this system at energies of 15 keV, 20 keV, 30 keV, and 37.7 keV were examined using a test object, and the measured values are shown to be in good agreement with theoretical values. High-energy x-ray nano-CT in combination with x-ray micro-CT is applied for 3D multiscale imaging. The entire bodies of bulky samples, ∼1 mm in diameter, were measured with the micro-CT, and the nano-CT was used for nondestructive observation of regions of interest. Examples of multiscale CT measurements involving carbon steel, mouse bones, and a meteorite are discussed.

Auditory ossicles in the middle ear and bony labyrinth of the inner ear are highly mineralized in adult mammals. Cellular mechanisms underlying formation of dense bone during development are unknown. Here, we found that osteoblast-like cells synthesizing highly mineralized hearing-related bones produce both type I and type II collagens as the bone matrix, while conventional osteoblasts and chondrocytes primarily produce type I and type II collagens, respectively. Furthermore, these osteoblast-like cells were not labeled in a “conventional osteoblast”-specific green fluorescent protein (GFP) mouse line. Type II collagen-producing osteoblast-like cells were not chondrocytes as they express osteocalcin, localize along alizarin-labeled osteoid, and form osteocyte lacunae and canaliculi, as do conventional osteoblasts. Auditory ossicles and the bony labyrinth exhibit not only higher bone matrix mineralization but also a higher degree of apatite orientation than do long bones. Therefore, we conclude that these type II collagen-producing hypermineralizing osteoblasts (termed here auditory osteoblasts) represent a new osteoblast subtype. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Objectives: The aim of this study is to investigate the underlying mechanism of the recovery of periodontal ligament cells (PDLCs) sequentially exposed to inflammation and mechanical loading. Materials and Methods: We divided PDLCs into four groups: control; compressive force (CF) alone (2.0 g/cm2); lipopolysaccharides (LPS) pretreatment (0.1 μg/ml) followed by simultaneous LPS and CF stimulation, simulating uncontrolled periodontitis; and LPS pretreatment followed by CF exposure, simulating controlled periodontitis. The expression of EphB4-ephrinB2 and EphA2-ephrinA2, and the level of osteoclastogenesis and osteogenesis were evaluated. Results: Simultaneous stimulation by LPS and CF, compared with CF alone and sequential LPS and CF exposure, significantly suppressed EphB4 and enhanced ephrinA2 expression. Similarly, the most intense osteoclastic differentiation was observed under simultaneous LPS and CF stimulation, while sequential exposure to LPS and CF only slightly increased osteoclastic cell numbers. Both the activation of EphB4 signaling and ephrinA2 silencing lowered osteoclastic differentiation, which had previously been upregulated by simultaneous LPS and CF stimulation. These treatments also increased osteogenic differentiation. Conclusions: Simultaneous LPS and CF stimulation critically enhances osteoclastogenesis in PDLCs through the suppression of EphB4 and the induction of ephrinA2 signaling. Sequential LPS and CF exposure partially abolishes the osteolytic effects of simultaneous stimulation.

Objective: Chordomas are low-grade but invasive tumors with limited therapies. Some conventional chordomas exhibit cartilage-like extracellular matrix (ECM). This study examined the expression and clinical significance of collagenases in chordomas. Methods: Brachyury-positive primary chordoma tissues were analyzed by immunohistochemistry for matrix metalloproteinase (MMP)13, MMP9, and cathepsin K, and by immunofluorescence for MMP13 and MMP9. ECM phenotype was categorized using safranin-O staining, where safranin-O marks cartilage-like ECM. We compared MMP13 expression scores and progression-free survival (PFS) between safranin-O-negative and -positive groups. Collagenase gene expression and protein localization in JHC7 cells were analyzed by quantitative PCR and immunocytochemistry, respectively. Collagen digestion activity was evaluated using fluorescein isothiocyanate–labeled type II collagen (COL2) in the presence or absence of an MMP13-specific inhibitor. Cell growth was evaluated in the presence of type I collagen (COL1) or COL2. Results: Safranin-O negative chordomas had shorter PFS than safranin-O positive chordomas (p = 0.016). MMP13 was expressed in human chordoma tissues and JHC7 cells; the MMP13 expression score was higher in safranin-O-negative chordomas than in safranin-O-positive chordomas (p = 0.018). JHC7 cells digested COL2, and digestion was partially but significantly inhibited by an MMP13-specific inhibitor (p < 0.05). COL2 inhibited the growth of JHC7 cells more strongly than COL1 in a dose-dependent manner (p < 0.01). Conclusions: MMP13 may promote aggressive behavior in chordoma by degrading growth-inhibitory COL2-rich ECM. These data support MMP13 as a potential unfavorable prognostic marker and therapeutic target in chordoma.

Tartrate-resistant acid phosphatase (TRAP) staining is widely used to stain osteoclasts in histological bone sections. The red dye formed by the conventional TRAP enzymatic reaction using naphthol AS-MX (or AS-BI) phosphate and fast red-violet (or garnet) chromogens is readily soluble in alcohol or xylene and requires air-drying prior to cover slipping or the use of an aqueous mounting medium. However, the use of an aqueous mounting medium makes it difficult to store stained specimens for a long time. In this modified method, a new fuchsin (NewFuchsin) was used as a chromogen, which enabled dehydration and clearing after staining and the use of a non-aqueous organic solvent-based mounting medium. Samples prepared using this modified TRAP activity staining method (NewFuchsin TRAP staining) have the following advantages over conventional TRAP staining: • The staining of sections provides a clear histological image and allows for long-term preservation. • The red dye formed by NewFuchsin TRAP staining can be detected not only in the bright field, but also in the fluorescent field. • Combined with tissue clearing using ethyl cinnamate, osteoclasts are observed using three-dimensional imaging.

The auditory brainstem response (ABR) is a scalp recording of potentials produced by sound stimulation, and is commonly used as an indicator of auditory function. However, the ABR threshold, which is the lowest audible sound pressure, cannot be objectively determined since it is determined visually using a measurer, and this has been a problem for several decades. Although various algorithms have been developed to objectively determine ABR thresholds, they remain lacking in terms of accuracy, efficiency, and convenience. Accordingly, we proposed an improved algorithm based on the mutual covariance at adjacent sound pressure levels. An ideal ABR waveform with clearly defined waves I–V was created; moreover, using this waveform as a standard template, the experimentally obtained ABR waveform was inspected for disturbances based on mutual covariance. The ABR testing was repeated if the value was below the established cross-covariance reference value. Our proposed method allowed more efficient objective determination of ABR thresholds and a smaller burden on experimental animals.

The year 2020 will be remembered for the coronavirus disease 2019 (COVID-19) pandemic, which continues to affect the whole world. Early and accurate identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is fundamental to combat the disease. Among the current diagnostic tests, real-time reverse transcriptase–polymerase chain reaction (RT-qPCR) is the most reliable and frequently used method. Herein, we discuss the interpretation of RT-qPCR results relative to viral infectivity. Although nasopharyngeal swab samples are often used for RT-qPCR testing, they require collection by trained medical staff. Saliva samples are emerging as an inexpensive and efficient alternative for large-scale screening. Pooled-sample testing of saliva has been applied for mass screening of SARS-CoV-2 infection. Current policies recommend isolating people with borderline cycle threshold (Ct) values (35

Bone marrow endothelial cells (BMECs) play a key role in bone formation and haematopoiesis. Although recent studies uncovered the cellular taxonomy of stromal compartments in the bone marrow (BM), the complexity of BMECs is not fully characterized. In the present study, using single-cell RNA sequencing, we defined a spatial heterogeneity of BMECs and identified a capillary subtype, termed type S (secondary ossification) endothelial cells (ECs), exclusively existing in the epiphysis. Type S ECs possessed unique phenotypic characteristics in terms of structure, plasticity and gene expression profiles. Genetic experiments showed that type S ECs atypically contributed to the acquisition of bone strength by secreting type I collagen, the most abundant bone matrix component. Moreover, these cells formed a distinct reservoir for haematopoietic stem cells. These findings provide the landscape for the cellular architecture in the BM vasculature and underscore the importance of epiphyseal ECs during bone and haematopoietic development.

The chemokine fractalkine (FKN) is produced by various cell types, including osteoblasts and endothelial cells in bone tissue, and signals through a sole receptor, CX3CR1, which is expressed on monocytes/macrophages, including osteoclast precursors (OCPs). However, the direct effects of FKN signaling on osteoclast lineage cells under homeostatic noninflammatory conditions remain unclear. Here, we report that FKN regulates mouse OCP survival and primes OCPs for subsequent osteoclast differentiation. Wild-type but not CX3CR1-deficient OCPs grown on immobilized FKN showed enhanced osteoclast formation following receptor activator of NF-κB ligand (RANKL) stimulation, with increased expression of osteoclast differentiation markers. Interestingly, the growth of OCPs on immobilized FKN increased the expression of Cx3cr1 and Tnfrsf11a (Rank) transcripts, but following RANKL stimulation, OCPs rapidly downregulated Cx3cr1 expression. Consistently, anti-FKN monoclonal antibody (mAb) treatment attenuated RANKL-induced osteoclast formation on immobilized FKN before, but not during, RANKL stimulation. CX3CR1 and RANK proteins were highly expressed on bone marrow-derived CD11bhigh CD115+ OCPs. Growth on immobilized FKN prior to RANKL stimulation also increased CD11bhigh CD115+ OCP number and their survival and differentiation potential. In a RANKL-based mouse model of bone loss, anti-FKN mAb pretreatment significantly inhibited RANKL-dependent bone loss. Thus, blocking the FKN-CX3CR1 axis could represent a therapeutic option in noninflammatory bone loss diseases. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Chirality is an intrinsic property of cells manifested as left-right (LR) asymmetry in terms of cellular morphology and organization, which influences cell behavior, migration, and tissue development. Traditional in vitro methods used to study cell chirality often require complex fabrication methods, limiting their accessibility and reproducibility. Here, we present a novel micropattern tape method that facilitates fabrication of high-quality rectangular micropatterns useful for efficient, high-throughput analysis of cell chirality. Using this method, we characterized chirality of C2C12 myoblasts and MC3T3-E1 osteoblasts, which respectively exhibit clockwise (CW) and counterclockwise (CCW) chirality relative to the long axis of the rectangle. We used the method to analyze how cellular differentiation impacts chirality and observed striking reversal of C2C12 cell chirality upon bone morphogenic protein-2 (BMP2)-induced osteoblastic differentiation. These results demonstrate that our micropattern tape method can effectively detect dynamic change of cell chirality during differentiation.

Background: During the COVID-19 pandemic, there was an increasing need to expand diagnostic testing in hospitals. At Keio University Hospital (KUH), clinical staff were concerned that the demand for PCR testing might exceed the capacity of the Clinical Laboratory. In response, basic researchers at Keio University School of Medicine (KUSM) set out to build a new, collaborative, PCR testing system. To be authorized to perform such diagnostic PCR testing, KUSM registered its core laboratory as an external clinical laboratory (ECL). Methods: In the pandemic, there was a pressure to build the PCR system quickly. Speed required discussions that developed a shared understanding of the unprecedented, new KUH/KUSM PCR system. To design, construct, and archive the new PCR testing system, we used a systems engineering (SE) approach. This included diagram visualization of functional flows and application of the Unified Architecture Framework (UAF), both of which are often used in system building. We considered daily demand for PCR testing at KUH and KUSM, and daily COVID-19 infections in Japan. Results: We operated the collaborative PCR testing system from August 2020 to June 2022. Given public health insurance reimbursement policies, KUH focused on individuals with suspicious symptoms, while the ECL at KUSM screened samples from asymptomatic individuals. KUSM performed about half as many tests as KUH. Interviewing KUH staff revealed that diagrams helped promote a better understanding of the KUH/KUSM PCR testing system. Conclusion: When designing temporary systems that may be repurposed in the future, we suggest using an SE approach with diagrams and UAF perspectives. This approach will enable stakeholders to understand what is being proposed to be built, and facilitate achieving an informed consensus on the proposed system. We suggest that SE approaches should be widely used in projects that involve building and operating complex, collaborative systems, and documenting the process.

Smoking is a common risk factor for both chronic obstructive pulmonary disease (COPD) and osteoporosis. In patients with COPD, severe emphysema is a risk factor for vertebral fracture; however, the effects of smoking or emphysema on bone health remain largely unknown. We report bone deterioration in a mouse model of emphysema induced by nose-only cigarette smoke (CS) exposure. Unexpectedly, short-term exposure for 4-weeks decreased bone turnover and increased bone volume in mice. However, prolonged exposure for 20- and 40-weeks reversed the effects from suppression to promotion of bone resorption. This long-term CS exposure increased osteoclast number and impaired bone growth, while it increased bone volume. Strikingly, long-term CS exposure deteriorated bone quality of the lumbar vertebrae as illustrated by disorientation of collagen fibers and the biological apatite c-axis. This animal model may provide a better understanding of the mechanisms underlying the deterioration of bone quality in pulmonary emphysema caused by smoking.