Fibroblast growth factor signalling regulates the development of tooth root.

Fibroblast growth factor (FGF) signalling plays a crucial role in the morphogenesis of multiple tissues including teeth. While the role of the signal has been studied in tooth crown development, little is known about root development. Of several FGF ligands involved in hard tissue formation, we suggest that FGF18 regulates the development of murine tooth roots. We implanted FGF18-soaked heparin beads into the lower first molar tooth buds at postnatal day 6 (P6), followed by transplantation under the kidney capsule. After 3 weeks, FGF18 significantly facilitated root elongation and periodontal tissue formation compared to the control. In situ hybridisation showed that Fgf18 transcripts were initially localised in the dental pulp along Hertwig's epithelial root sheath at P6 and P10 and subsequently in the dental follicle cells at P14. Fgf receptors were expressed in various dental tissues during these stages. In vitro analysis using the dental pulp stem cells revealed that FGF18 inhibited cell proliferation and decreased expression levels of osteogenic markers, Runx2, Alpl and Sp7. Consistently, after 1 week of kidney capsule transplantation, FGF18 application did not induce the expression of Sp7 and Bsp, but upregulated Periostin in the apical region of dental mesenchyme in the grafted molar. These findings suggest that FGF18 facilitates molar root development by regulating the calcification of periodontal tissues.

In-process sintering of Au nanoparticles deposited in laser-assisted electrophoretic deposition.

In this study, we developed an in-process sintering method for laser-assisted electrophoretic deposition (LAEPD) using an additional laser to sinter Au particles and improve the Young's modulus of the microstructures fabricated using LAEPD. Thus, in addition to the laser (λ = 488 nm) that traps nanoparticles, another laser (λ = 785 nm) was installed to effectively absorb and sinter the deposited nanoparticles. Deposition was performed via LAEPD and laser sintering alternatively during fabrication. A Young's modulus of 28.2 GPa was achieved for the Au pillar fabricated with a sintering laser irradiation time of 1000 ms/cycle.

Remnant tissue enhances early postoperative biomechanical strength and infiltration of Scleraxis-positive cells within the grafted tendon in a rat anterior cruciate ligament reconstruction model.

When ruptured, ligaments and tendons have limited self-repair capacity and rarely heal spontaneously. In the knee, the Anterior Cruciate Ligament (ACL) often ruptures during sports activities, causing functional impairment and requiring surgery using tendon grafts. Patients with insufficient time to recover before resuming sports risk re-injury. To develop more effective treatment, it is necessary to define mechanisms underlying ligament repair. For this, animal models can be useful, but mice are too small to create an ACL reconstruction model. Thus, we developed a transgenic rat model using control elements of Scleraxis (Scx), a transcription factor essential for ligament and tendon development, to drive GFP expression in order to localize Scx-expressing cells. As anticipated, Tg rats exhibited Scx-GFP in ACL during developmental but not adult stages. Interestingly, when we transplanted the flexor digitorum longus (FDP) tendon derived from adult Scx-GFP+ rats into WT adults, Scx-GFP was not expressed in transplanted tendons. However, tendons transplanted from adult WT rats into Scx-GFP rats showed upregulated Scx expression in tendon, suggesting that Scx-GFP+ cells are mobilized from tissues outside the tendon. Importantly, at 4 weeks post-surgery, Scx-GFP-expressing cells were more frequent within the grafted tendon when an ACL remnant was preserved (P group) relative to when it was not (R group) (P vs R groups (both n = 5), p<0.05), and by 6 weeks, biomechanical strength of the transplanted tendon was significantly increased if the remnant was preserved (P vsR groups (both n = 14), p<0.05). Scx-GFP+ cells increased in remnant tissue after surgery, suggesting remnant tissue is a source of Scx+ cells in grafted tendons. We conclude that the novel Scx-GFP Tg rat is useful to monitor emergence of Scx-positive cells, which likely contribute to increased graft strength after ACL reconstruction.

Augmented effect of fibroblast growth factor 18 in bone morphogenetic protein 2-induced calvarial bone healing by activation of CCL2/CCR2 axis on M2 macrophage polarization.

Fibroblast growth factor (FGF) signaling plays essential roles in various biological events. FGF18 is one of the ligands to be associated with osteogenesis, chondrogenesis and bone healing. The mouse critical-sized calvarial defect healing induced by the bone morphogenetic protein 2 (BMP2)-hydrogel is stabilized when FGF18 is added. Here, we aimed to investigate the role of FGF18 in the calvarial bone healing model. We first found that FGF18 + BMP2 hydrogel application to the calvarial bone defect increased the expression of anti-inflammatory markers, including those related to tissue healing M2 macrophage (M2-Mø) prior to mineralized bone formation. The depletion of macrophages with clodronate liposome hindered the FGF18 effect. We then examined how FGF18 induces M2-Mø polarization by using mouse primary bone marrow (BM) cells composed of macrophage precursors and BM stromal cells (BMSCs). In vitro studies demonstrated that FGF18 indirectly induces M2-Mø polarization by affecting BMSCs. Whole transcriptome analysis and neutralizing antibody treatment of BMSC cultured with FGF18 revealed that chemoattractant chemokine (c-c motif) ligand 2 (CCL2) is the major mediator for M2-Mø polarization. Finally, FGF18-augmented activity toward favorable bone healing with BMP2 was diminished in the calvarial defect in Ccr2-deleted mice. Altogether, we suggest a novel role of FGF18 in M2-Mø modulation via stimulation of CCL2 production in calvarial bone healing.

Potential function of Scx+/Sox9+ cells as progenitor cells in rotator cuff tear repair in rats.

Tendons and their attachment sites to bone, fibrocartilaginous tissues, have poor self-repair capacity when they rupture, and have risks of retear even after surgical repair. Thus, defining mechanisms underlying their repair is required in order to stimulate tendon repairing capacity. Here we used a rat surgical rotator cuff tear repair model and identified cells expressing the transcription factors Scleraxis (Scx) and SRY-box 9 (Sox9) as playing a crucial role in rotator cuff tendon-to-bone repair. Given the challenges of establishing stably reproducible models of surgical rotator cuff tear repair in mice, we newly established Scx-GFP transgenic rats in which Scx expression can be monitored by GFP. We observed tissue-specific GFP expression along tendons in developing ScxGFP transgenic rats and were able to successfully monitor tissue-specific Scx expression based on GFP signals. Among 3-, 6-, and 12-week-old ScxGFP rats, Scx+/Sox9+ cells were most abundant in 3-week-old rats near the site of humerus bone attachment to the rotator cuff tendon, while we observed significantly fewer cells in the same area in 6- or 12-week-old rats. We then applied a rotator cuff repair model using ScxGFP rats and observed the largest number of Scx+/Sox9+ cells at postoperative repair sites of 3-week-old relative to 6- or 12-week-old rats. Tendons attach to bone via fibrocartilaginous tissue, and cartilage-like tissue was seen at repair sites of 3-week-old but not 6- or 12-week-old rats during postoperative evaluation. Our findings suggest that Scx+/Sox9+ cells may function in rotator cuff repair, and that ScxGFP rats could serve as useful tools to develop therapies to promote rotator cuff repair by enabling analysis of these activities.

Digits in a dish: An in vitro system to assess the molecular genetics of hand/foot development at single-cell resolution.

In vitro models allow for the study of developmental processes outside of the embryo. To gain access to the cells mediating digit and joint development, we identified a unique property of undifferentiated mesenchyme isolated from the distal early autopod to autonomously re-assemble forming multiple autopod structures including: digits, interdigital tissues, joints, muscles and tendons. Single-cell transcriptomic analysis of these developing structures revealed distinct cell clusters that express canonical markers of distal limb development including: Col2a1, Col10a1, and Sp7 (phalanx formation), Thbs2 and Col1a1 (perichondrium), Gdf5, Wnt5a, and Jun (joint interzone), Aldh1a2 and Msx1 (interdigital tissues), Myod1 (muscle progenitors), Prg4 (articular perichondrium/articular cartilage), and Scx and Tnmd (tenocytes/tendons). Analysis of the gene expression patterns for these signature genes indicates that developmental timing and tissue-specific localization were also recapitulated in a manner similar to the initiation and maturation of the developing murine autopod. Finally, the in vitro digit system also recapitulates congenital malformations associated with genetic mutations as in vitro cultures of Hoxa13 mutant mesenchyme produced defects present in Hoxa13 mutant autopods including digit fusions, reduced phalangeal segment numbers, and poor mesenchymal condensation. These findings demonstrate the robustness of the in vitro digit system to recapitulate digit and joint development. As an in vitro model of murine digit and joint development, this innovative system will provide access to the developing limb tissues facilitating studies to discern how digit and articular joint formation is initiated and how undifferentiated mesenchyme is patterned to establish individual digit morphologies. The in vitro digit system also provides a platform to rapidly evaluate treatments aimed at stimulating the repair or regeneration of mammalian digits impacted by congenital malformation, injury, or disease.

Mechanisms of skeletal muscle-tendon development and regeneration/healing as potential therapeutic targets.

Skeletal muscle contraction is essential for the movement of our musculoskeletal system. Tendons and ligaments that connect the skeletal muscles to bones in the correct position at the appropriate time during development are also required for movement to occur. Since the musculoskeletal system is essential for maintaining basic bodily functions as well as enabling interactions with the environment, dysfunctions of these tissues due to disease can significantly reduce quality of life. Unfortunately, as people live longer, skeletal muscle and tendon/ligament diseases are becoming more common. Sarcopenia, a disease in which skeletal muscle function declines, and tendinopathy, which involves chronic tendon dysfunction, are particularly troublesome because there have been no significant advances in their treatment. In this review, we will summarize previous reports on the development and regeneration/healing of skeletal muscle and tendon tissues, including a discussion of the molecular and cellular mechanisms involved that may be used as potential therapeutic targets.

Effect of a Bifidobacterium-Containing Acid-Resistant Microcapsule Formulation on Gut Microbiota: A Pilot Study.

Approximately 10 Bifidobacterium species are known to inhabit the human intestinal tract. Bifidobacteria have been reported to possess a variety of probiotic benefits. However, when bifidobacteria are consumed internally as probiotics, the bacteria are killed by gastric acid. Therefore, we developed acid-resistant microcapsules containing Bifidobacterium breve M-16V and B. longum BB536, which are unaffected by gastric acid, and evaluated whether the microcapsule formulation increased the amount of bifidobacteria in the stool after administration compared with the powder formulation. The results revealed no significant difference in the percentage or number of B. longum between before and after administration of the powder or microcapsule formulation in children. By contrast, the bacterial count of B. breve was significantly increased after microcapsule formulation administration (1.5 × 105 copies/g after administration versus 2.8 × 104 copies/g before administration, p = 0.013). In addition, the increase in the bacterial count of B. breve in stools after administration of microcapsule formulation was approximately 1000-fold higher than that after powder formulation administration (p = 0.018). In conclusion, the results indicate that the microcapsule formulation is efficiently transferred to the large intestine without the adverse effects of gastric acidity in children.

Pancreaticoenterostomy With Seromuscular-parenchymal Anastomosis for Prevention of Postoperative Pancreatic Fistula in Distal Pancreatectomy.

BACKGROUND/AIM: There have been several attempts to prevent the development of a postoperative pancreatic fistula (POPF) after distal pancreatectomy (DP); however, there is no established method yet. In the present study, we investigated whether POPF can be prevented using pancreaticoenterostomy with seromuscular-parenchymal anastomosis. PATIENTS AND METHODS: We evaluated the incidence of POPF and complications in 20 patients who underwent DP since August 2014, wherein pancreaticoenterostomy with seromuscular-parenchymal anastomosis was performed. RESULTS: No patient developed POPF, and only 4 patients developed a biochemical leak. Postoperative complications (Clavien-Dindo classification: CD) occurred in 5 patients (Grade IIIa in 2 cases, Grade II in 2 cases, and Grade I in 1 case). In a case of CD Grade II, a gastric ulcer was formed at the pancreatico-gastric anastomosis. CONCLUSION: Although new complications, such as the formation of ulcers, consistent with pancreatic anastomosis, were noted, the present method was useful in preventing POPF.

Septic shock in a woman with a hydatidiform mole: A case report.

Hydatidiform moles can be fatal because of the risk of massive bleeding or thyroid storm; however, they rarely occur concomitantly with sepsis. We present herein the case of a woman with a hydatidiform mole with septic shock. A 30-year-old multiparous woman with Basedow's disease presented with fever, amenorrhea, and vaginal bleeding. Transvaginal ultrasonography revealed an enlarged uterus with an intrauterine vesicular mass (74.3 × 93.0 mm). Human chorionic gonadotropin level was 994,000 mIU/mL. C-reactive protein was elevated, and blood cultures were positive (gram-negative rods), indicating infection. After administering antibiotics (tazobactam and piperacillin), blood pressure suddenly decreased (69/45 mmHg), requiring stabilization with noradrenaline and albumin. The uterine contents were naturally expelled, followed by dilatation and curettage after her vital signs and general condition gradually improved. The pathological diagnosis was a complete hydatidiform mole. Culture of the intrauterine contents revealed Escherichia coli, leading to the potentially fatal diagnosis of septic shock associated with a hydatidiform mole.

Computational Design to Suppress Thermal Runaway of Li-Ion Batteries via Atomic Substitutions to Cathode Materials.

The cathode material of a lithium-ion battery is a key component that affects durability, capacity, and safety. Compared to the LiCoO2 cathode material (the reference standard for these properties), LiNiO2 can extract more Li at the same voltage and has therefore attracted considerable attention as a material that can be used to obtain higher capacity. As a trade-off, it undergoes pyrolysis relatively easily, leading to ignition and explosion hazards, which is a challenge associated with the application of this compound. Pyrolysis has been identified as a structural phase transformation of the layered rocksalt structure → spinel → cubic rocksalt. Partial substitution of Ni with various elements can reportedly suppress the transformation and, hence, the pyrolysis. It remains unclear which elemental substitutions inhibit pyrolysis and by what mechanism, leading to costly material development that relies on empirical trial and error. In this study, we developed several possible reaction models based on existing reports, estimated the enthalpy change associated with the reaction by ab initio calculations, and identified promising elemental substitutions. The possible models were narrowed down by analyzing the correlations of the predicted dependence of the reaction enthalpies on elemental substitutions, compared between different reaction models. According to this model, substitution by P and Ta affords the highest enthalpy barrier between the initial (layered rocksalt) and the final (cubic rocksalt) structures but promotes the initial transformation to spinel as a degradation. Substitution by W instead generates the barrier to the final (preventing dangerous incidents) process, as well as for the initial degradation to spinel; therefore, it is a promising strategy to suppress the predicted pyrolysis.

Potential critical risks of pulmonary thromboembolism from an asymptomatic postpartum ovarian vein thrombosis: a case report.

BACKGROUND: Ovarian vein thrombosis (OVT) may cause maternal mortality by inducing pulmonary thromboembolism (PTE). However, the prevalence, etiology, risk factors, prognosis, and optimal treatments for asymptomatic OVT during and after pregnancies are unclear, which therefore requires a high clinical index of suspicion for certain diagnoses due to its vague presentation. We herein present a case of asymptomatic postpartum OVT that extended toward the inferior vena cava (IVC), resulting in a potential risk of PTE. CASE PRESENTATION: A 30-year-old postpartum woman presented with slight dyspnea after an uneventful vaginal delivery at 40 weeks of gestation. We checked her laboratory data to exclude lethal thrombosis; D-dimer levels were 85.6 µg/mL. We performed computed tomography (CT) to search the presence of PTE and deep vein thrombosis (DVT); although no signs of PTE and DVT in her legs were detected, CT and trans-abdominal ultrasonography (TAUS) revealed a right OVT. Heparin was administered, and D-dimer levels decreased; warfarin at a dose of 2 mg/day was subsequently administered to control anti-coagulopathy. However, D-dimer was re-elevated despite adequate anticoagulation treatment, and extension of the right OVT to the IVC was detected by CT and TAUS. With warfarin administration, CT and TAUS showed the disappearance of right OVT. The patient was discharged from the hospital 17 days after delivery. CONCLUSIONS: Even asymptomatic postpartum OVT may lead to PTE. Universal screening guidelines and optimal treatment strategies for asymptomatic OVT in pregnant and postpartum women should be established through future studies.

Tenogenic Induction From Induced Pluripotent Stem Cells Unveils the Trajectory Towards Tenocyte Differentiation.

The musculoskeletal system is integrated by tendons that are characterized by the expression of scleraxis (Scx), a functionally important transcription factor. Here, we newly developed a tenocyte induction method using induced pluripotent stem cells established from ScxGFP transgenic mice by monitoring fluorescence, which reflects a dynamic differentiation process. Among several developmentally relevant factors, transforming growth factor-beta 2 (TGF-ß2) was the most potent inducer for differentiation of tenomodulin-expressing mature tenocytes. Single-cell RNA sequencing (scRNA-seq) revealed 11 distinct clusters, including mature tenocyte population and tenogenic differentiation trajectory, which recapitulated the in vivo developmental process. Analysis of the scRNA-seq dataset highlighted the importance of retinoic acid (RA) as a regulatory pathway of tenogenic differentiation. RA signaling was shown to have inhibitory effects on entheseal chondrogenic differentiation as well as TGF-ß2-dependent tenogenic/fibrochondrogenic differentiation. The collective findings provide a new opportunity for tendon research and further insight into the mechanistic understanding of the differentiation pathway to a tenogenic fate.

Gallbladder wall thickening in a woman with postpartum preeclampsia: A case report.

BACKGROUND: Preeclampsia (PE) is hallmarked by dysfunction of various organs; therefore, its diagnosis can be challenging, especially when patients present with right upper abdominal pain. Herein, we present a case of postpartum gallbladder wall thickening (GBWT) that led to a diagnosis of PE, rather than hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome or gallbladder and biliary disease. CASE PRESENTATION: A 31-year-old postpartum woman presented with a fever, hypertension, headache, and right upper abdominal pain. HELLP syndrome and intracranial hemorrhage were initially suspected, due to the combination of symptoms and elevated levels of aspartate transaminase, alanine transaminase, and lactate dehydrogenase. However, hemolysis and thrombocytopenia were absent, and a computed tomography (CT) scan of the head did not indicate the presence of intracranial hemorrhage. Further, transabdominal ultrasound and CT revealed GBWT (edematous gallbladder); CT also revealed an enlarged heart, lung edema, pleural effusion, and ascites. Thus, PE, rather than HELLP syndrome or gallbladder or biliary disease, was diagnosed based on gestational hypertension and proteinuria, new-onset headache, liver dysfunction, and edema in several organs, including the lung. Nicardipine treatment quickly improved hypertension and headache, and, over time, the patient's urination increased, and edema subsided throughout the body. Furthermore, laboratory results improved, and the patient was discharged on postpartum day 11. CONCLUSION: Postpartum gallbladder wall thickening can be a diagnostic sign of PE.

Increased MFG-E8 at neuromuscular junctions is an exacerbating factor for sarcopenia-associated denervation.

Sarcopenia is an important health problem associated with adverse outcomes. Although the etiology of sarcopenia remains poorly understood, factors apart from muscle fibers, including humoral factors, might be involved. Here, we used cytokine antibody arrays to identify humoral factors involved in sarcopenia and found a significant increase in levels of milk fat globule epidermal growth factor 8 (MFG-E8) in skeletal muscle of aged mice, compared with young mice. We found that the increase in MFG-E8 protein at arterial walls and neuromuscular junctions (NMJs) in muscles of aged mice. High levels of MFG-E8 at NMJs and an age-related increase in arterial MFG-E8 have also been identified in human skeletal muscle. In NMJs, MFG-E8 is localized on the surface of terminal Schwann cells, which are important accessory cells for the maintenance of NMJs. We found that increased MFG-E8 at NMJs precedes age-related denervation and is more prominent in sarcopenia-susceptible fast-twitch than in sarcopenia-resistant slow-twitch muscle. Comparison between fast and slow muscles further revealed that arterial MFG-E8 can be uncoupled from sarcopenic phenotype. A genetic deficiency in MFG-E8 attenuated age-related denervation of NMJs and muscle weakness, providing evidence of a pathogenic role of increased MFG-E8. Thus, our study revealed a mechanism by which increased MFG-E8 at NMJs leads to age-related NMJ degeneration and suggests that targeting MFG-E8 could be a promising therapeutic approach to prevent sarcopenia.

Measurement of Lateral Transmission of Force in the Extensor Digitorum Longus Muscle of Young and Old Mice.

The main function of skeletal muscles is to generate force. The force developed by myofiber contraction is transmitted to the tendon. There are two pathways of force transmission from myofibers to tendons: longitudinal transmission that depends on tension elicited via the myotendinous junction and lateral transmission that depends on shear elicited via the interface between the myofiber surface and surrounding connective tissue. Experiments using animal muscle and mathematical models indicated that lateral transmission is the dominant pathway in muscle force transmission. Studies using rat muscle showed that the efficiency of lateral force transmission declines with age. Here, the lateral transmission of force was measured using the extensor digitorum longus muscle from young and old mice. Dependence on longitudinal transmission increased in the old muscle, and there was a trend for lower efficiency of lateral force transmission in the old muscle compared to the young muscle. There was a noticeable increase in the connective tissue volume in the old muscle; however, there was no significant change in the expression of dystrophin, a critical molecule for the link between the myofiber cytoskeleton and extracellular matrix. This study demonstrates the measurement of lateral force transmission in mouse muscles and that alteration in force transmission property may underlie age-related muscle weakness.

Scleraxis upregulated by transforming growth factor-ß1 signaling inhibits tension-induced osteoblast differentiation of priodontal ligament cells via ephrin A2.

During tooth movement in orthodontic treatment, bone formation and resorption occur on the tension and compression sides of the alveolar bone, respectively. Although the bone formation activity increases in the periodontal ligament (PDL) on the tension side, the PDL itself is not ossified and maintains its homeostasis, indicating that there are negative regulators of bone formation in the PDL. Our previous report suggested that scleraxis (Scx) has an inhibitory effect on ossification of the PDL on the tension side through the suppression of calcified extracellular matrix formation. However, the molecular biological mechanisms of Scx-modulated inhibition of ossification in the tensioned PDL are not fully understood. The aim of the present study is to clarify the inhibitory role of Scx in osteoblast differentiation of PDL cells and its underlying mechanism. Our in vivo experiment using a mouse experimental tooth movement model showed that Scx expression was increased during early response of the PDL to tensile force. Scx knockdown upregulated expression of alkaline phosphatase, an early osteoblast differentiation marker, in the tensile force-loaded PDL cells in vitro. Transforming growth factor (TGF)-ß1-Smad3 signaling in the PDL was activated by tensile force and inhibitors of TGF-ß receptor and Smad3 suppressed the tensile force-induced Scx expression in PDL cells. Tensile force induced ephrin A2 (Efna2) expression in the PDL and Efna2 knockdown upregulated alkaline phosphatase expression in PDL cells under tensile force loading. Scx knockdown eliminated the tensile force-induced Efna2 expression in PDL cells. These findings suggest that the TGF-ß1-Scx-Efna2 axis is a novel molecular mechanism that negatively regulates the tensile force-induced osteoblast differentiation of PDL cells.

Mesenchymal Bmp3b expression maintains skeletal muscle integrity and decreases in age-related sarcopenia.

Age-related sarcopenia constitutes an important health problem associated with adverse outcomes. Sarcopenia is closely associated with fat infiltration in muscle, which is attributable to interstitial mesenchymal progenitors. Mesenchymal progenitors are nonmyogenic in nature but are required for homeostatic muscle maintenance. However, the underlying mechanism of mesenchymal progenitor-dependent muscle maintenance is not clear, nor is the precise role of mesenchymal progenitors in sarcopenia. Here, we show that mice genetically engineered to specifically deplete mesenchymal progenitors exhibited phenotypes markedly similar to sarcopenia, including muscle weakness, myofiber atrophy, alterations of fiber types, and denervation at neuromuscular junctions. Through searching for genes responsible for mesenchymal progenitor-dependent muscle maintenance, we found that Bmp3b is specifically expressed in mesenchymal progenitors, whereas its expression level is significantly decreased during aging or adipogenic differentiation. The functional importance of BMP3B in maintaining myofiber mass as well as muscle-nerve interaction was demonstrated using knockout mice and cultured cells treated with BMP3B. Furthermore, the administration of recombinant BMP3B in aged mice reversed their sarcopenic phenotypes. These results reveal previously unrecognized mechanisms by which the mesenchymal progenitors ensure muscle integrity and suggest that age-related changes in mesenchymal progenitors have a considerable impact on the development of sarcopenia.

Role of Scx+/Sox9+ cells as potential progenitor cells for postnatal supraspinatus enthesis formation and healing after injury in mice.

A multipotent cell population co-expressing a basic-helix-loop-helix transcription factor scleraxis (Scx) and SRY-box 9 (Sox9) has been shown to contribute to the establishment of entheses (tendon attachment sites) during mouse embryonic development. The present study aimed to investigate the involvement of Scx+/Sox9+ cells in the postnatal formation of fibrocartilaginous entheses and in the healing process after injury, using ScxGFP transgenic mice. We demonstrate that Scx+/Sox9+ cells are localized in layers at the insertion site during the postnatal formation of fibrocartilaginous entheses of supraspinatus tendon until postnatal 3 weeks. Further, these cells were rarely seen at postnatal 6 weeks, when mature fibrocartilaginous entheses were formed. Furthermore, we investigated the involvement of Scx+/Sox9+ cells in the healing process after supraspinatus tendon enthesis injury, comparing the responses of 20- and 3-week-old mice. In the healing process of 20-week-old mice with disorganized fibrovascular tissue in response to injury, a small number of Scx+/Sox9+ cells transiently appeared from 1 week after injury, but they were rarely seen at 4 weeks after injury. Meanwhile, in 3-week-old mice, a thin layer of fibrocartilaginous tissue with calcification was formed at healing enthesis at 4 weeks after injury. From 1 to 2 weeks after injury, more Scx+/Sox9+ cells, widely distributed at the injured site, were seen compared with the 20-week-old mice. At 4 weeks after injury, these cells were located near the surface of the recreated fibrocartilaginous layer. This spatiotemporal localization pattern of Scx+/Sox9+ cells at the injured enthesis in our 3-week-old mouse model was similar to that in postnatal fibrocartilaginous enthesis formation. These findings indicate that Scx+/Sox9+ cells may have a role as entheseal progenitor-like cells during postnatal maturation of fibrocartilaginous entheses and healing after injury in a manner similar to that seen in embryonic development.

Transient and lineage-restricted requirement of Ebf3 for sternum ossification.

Osteoblasts arise from bone-surrounding connective tissue containing tenocytes and fibroblasts. Lineages of these cell populations and mechanisms of their differentiation are not well understood. Screening enhancer-trap lines of zebrafish allowed us to identify Ebf3 as a transcription factor marking tenocytes and connective tissue cells in skeletal muscle of embryos. Knockout of Ebf3 in mice had no effect on chondrogenesis but led to sternum ossification defects as a result of defective generation of Runx2+ pre-osteoblasts. Conditional and temporal Ebf3 knockout mice revealed requirements of Ebf3 in the lateral plate mesenchyme cells (LPMs), especially in tendon/muscle connective tissue cells, and a stage-specific Ebf3 requirement at embryonic day 9.5-10.5. Upregulated expression of connective tissue markers, such as Egr1/2 and Osr1, increased number of Islet1+ mesenchyme cells, and downregulation of gene expression of the Runx2 regulator Shox2 in Ebf3-deleted thoracic LPMs suggest crucial roles of Ebf3 in the onset of lateral plate mesoderm differentiation towards osteoblasts forming sternum tissues.