Common and uncommon findings in marmoset and human trophoblast stem cells.

In this issue of Cell Stem Cell, Siriwardena et al. analyze peri- and post-implantation marmoset trophoblast development in detail and establish marmoset trophoblast stem cell (TSC) lines from pluripotent stem cells (PSCs). Comparative analysis of marmoset and human TSCs provides insights into species-specific implantation and placentation strategies.

Risk of self-harm ideation in mothers of children with orofacial cleft defects: the Japan environment and children's study.

Introduction: Cleft lip and/or palate (CL/P), the most prevalent congenital anomaly, has been associated with higher rates of child maltreatment. In particular, the presence of cleft lip has more of an impact on external appearance and may increase the risks of negative health outcomes such as parental postpartum depression; however, this concept remains controversial. Item #10 of the Edinburgh Postpartum Depression Scale is the assessment of parental self-harm ideation, and its presence in postpartum mothers merits risk assessments as an emergent issue that may affect the health of both mothers and infants. This study focused on the impact of CL/P on maternal self-harm ideation. Methods: Of 100,300 live births from a nationwide birth cohort in Japan, 238 mothers of infants with CL/P [186 children born with cleft lip (CL ± P) and 52 born with isolated cleft palate (CP)] were included in the analyses. The prospective association of children with CL/P and maternal self-harm ideation, which were acquired using item #10 in the Edinburgh Postpartum Depression Scale at 1 and 6 months postpartum, was examined using binomial logistic regression analyses after multiple imputations and with adjustments for several maternal (age at delivery, smoking habit, and alcohol intake) and child-related (sex and prevalence of other congenital diseases) variables. Results: The prevalence of self-harm ideation in 238 mothers of infants with CL/P at 1 and 6 months were 14.7% (35/238) and 18.8% (45/238) [8.2% (8,185/100,062) and 12.9% (12,875/100,062) in the control group], respectively. The odds ratio (95% confidence interval) for maternal self-harm ideation increased with CL/P prevalence [1.80 (1.22-2.65) and 1.47 (0.98-2.18)] at 1 and 6 months of age, respectively. After stratified by the prevalence of cleft lip, we found significant differences in the CL ± P group but not in the CP group. Furthermore, persistent self-harming ideation was associated with a higher risk in the CL ± P group [2.36 (1.43-3.89)]. Conclusion: CL/P, particularly cleft lip, which is more noticeable externally, was associated with an increased prevalence of maternal self-harm ideation. The findings in this study indicate some potential benefits of increasing support for mothers who have infants with CL/P.

EFFECTS OF ARYL HYDROCARBON RECEPTOR LIGAND TCDD ON HUMAN TROPHOBLAST CELL DEVELOPMENT.

BACKGROUND: The primary interface between mother and fetus, the placenta, serves two critical functions: extraction of nutrients from the maternal compartment and facilitation of nutrient delivery to the developing fetus. This delivery system also serves as a barrier to environmental exposures. The aryl hydrocarbon receptor (AHR) is an important component of the barrier. AHR signaling is activated by environmental pollutants and toxicants that can potentially affect cellular and molecular processes, including those controlling trophoblast cell development and function. OBJECTIVES: In this study, we investigated the impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an effective AHR ligand, exposure on human trophoblast cells. METHODS: Human trophoblast stem (TS) cells were used as in vitro model system for investigating the downstream consequences of AHR activation. The actions TCDD were investigated in human TS cells maintained in the stem state or in differentiating TS cells. RESULTS: TCDD exposure stimulated the expression of CYP1A1 and CYP1B1 in human TS cells. TCDD was effective in stimulating CYP1A1 and CYP1B1 expression and altering gene expression profiles in human TS cells maintained in the stem cell state or induced to differentiate into extravillous trophoblast cells (EVT) or syncytiotrophoblast (ST). These actions were dependent upon the presence of AHR. TCDD exposure did not adversely affect maintenance of the TS cell stem state or the ability of TS cells to differentiate into EVT cells or ST. However, TCDD exposure did promote the biosynthesis of 2 methoxy estradiol (2ME), a biologically active catechol estrogen, with the potential to modify the maternal-fetal interface. DISCUSSION: Human trophoblast cell responses to TCDD were dependent upon AHR signaling and possessed the potential to shape development and function of the human placentation site.

NOTUM-mediated WNT silencing drives extravillous trophoblast cell lineage development.

Trophoblast stem (TS) cells have the unique capacity to differentiate into specialized cell types, including extravillous trophoblast (EVT) cells. EVT cells invade into and transform the uterus where they act to remodel the vasculature facilitating the redirection of maternal nutrients to the developing fetus. Disruptions in EVT cell development and function are at the core of pregnancy-related disease. WNT-activated signal transduction is a conserved regulator of morphogenesis of many organ systems, including the placenta. In human TS cells, activation of canonical WNT signaling is critical for maintenance of the TS cell stem state and its downregulation accompanies EVT cell differentiation. We show that aberrant WNT signaling undermines EVT cell differentiation. Notum, palmitoleoyl-protein carboxylesterase (NOTUM), a negative regulator of canonical WNT signaling, was prominently expressed in first-trimester EVT cells developing in situ and up-regulated in EVT cells derived from human TS cells. Furthermore, NOTUM was required for optimal human TS cell differentiation to EVT cells. Activation of NOTUM in EVT cells is driven, at least in part, by endothelial Per-Arnt-Sim (PAS) domain 1 (also called hypoxia-inducible factor 2 alpha). Collectively, our findings indicate that canonical Wingless-related integration site (WNT) signaling is essential for maintenance of human trophoblast cell stemness and regulation of human TS cell differentiation. Downregulation of canonical WNT signaling via the actions of NOTUM is required for optimal EVT cell differentiation.

Leveraging chorionic villus biopsies for the derivation of patient-specific trophoblast stem cells.

Human trophoblast stem (TS) cells are an informative in vitro model for the generation and testing of biologically meaningful hypotheses. The goal of this project was to derive patient-specific TS cell lines from clinically available chorionic villus sampling biopsies. Cell outgrowths were captured from human chorionic villus tissue specimens cultured in modified human TS cell medium. Cell colonies emerged early during the culture and cell lines were established and passaged for several generations. Karyotypes of the newly established chorionic villus-derived trophoblast stem (TS CV ) cell lines were determined and compared to initial genetic diagnoses from freshly isolated chorionic villi. Phenotypes of TSCV cells in the stem state and following differentiation were compared to cytotrophoblast-derived TS (TS CT ) cells. TSCV and TSCT cells uniformly exhibited similarities in the stem state and following differentiation into syncytiotrophoblast and extravillous trophoblast cells. Chorionic villus tissue specimens provide a valuable source for TS cell derivation. They expand the genetic diversity of available TS cells and are associated with defined clinical outcomes. TSCV cell lines provide a new set of experimental tools for investigating trophoblast cell lineage development.

Association of maternal blood and umbilical cord blood plasma fatty acid levels with the body size at birth of Japanese infants.

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), mainly obtained from fish, have been implicated in fetal development. Because few studies have examined maternal and umbilical cord blood fatty acid levels and infant body size in Japan with a fish-eating culture, we examined differences in plasma fatty acid levels in pregnant women and infant size at birth. This study is a large birth cohort study of 1476 pairs of Japanese pregnant women and their infants. Maternal blood DHA levels and infant birth weight showed a positive relationship. However, analysis adjusted for gestational age did not reveal correlations. Negative relationships were found between cord blood DHA levels and infant body size, and between the difference in mother-to-child DHA levels and infant body size. Thus, the smaller the birth size, the higher the differences in umbilical cord blood DHA levels and mother-to-child DHA levels when considering gestational age.

Association between maternal physical activity from pre-pregnancy to child-rearing and their children's physical activity in early childhood among Japanese.

INTRODUCTION: This study aimed to determine the association between cumulative maternal physical activity level and their children's physical activity in early childhood. We also compared the influence of each maternal physical activity on children's physical activity in early childhood. METHODS: We analyzed the data from 1,067 Japanese mother-child pairs. Maternal physical activity was assessed using the International Physical Activity Questionnaire. Cumulative physical activity level in mothers was computed based on the categories (low, moderate, and high) of physical activity from 5 time points (pre-pregnancy, during pregnancy, 1.5, 3.5, and 5.5 years postpartum). Children's physical activity level was measured at age 5.5 years using the WHO Health Behaviour School-aged Children questionnaire and defined as engaging in physical activity for at least 60 minutes per day for more than 5 days. Logistic regression analysis was used to determine the association between maternal and children's physical activity levels. RESULTS: The results showed the positive association between cumulative maternal physical activity and children's physical activity level (P for trend < 0.001). Furthermore, maternal physical activity during pregnancy (P for trend = 0.031) and 5.5 years postpartum (P for trend < 0.001) was positively associated with children's physical activity. CONCLUSION: A positive association was observed between the cumulative maternal physical activity level and the physical activity level of their children at 5.5 years of age. Furthermore, maternal physical activity during pregnancy and at 5.5 years postpartum were positively associated with the level of children's physical activity.

Maternal birth weight is an indicator of preterm delivery: the Japan environment and children's study.

This study aimed to investigate the association between maternal birth weight (MBW) with preterm delivery (PTD) in the Japanese population. To this end, a total of 78,972 Japanese pregnant women were included in a prospective birth cohort study. Multiple logistic regression and multinominal logistic regression models were applied to investigate the associations of MBW with PTD (delivery from 22 to < 37 weeks of gestation), early PTD (delivery from 22 to < 34 weeks), and late PTD (delivery from 34 to < 37 weeks). The results showed that MBW was inversely associated with PTD, early PTD, and late PTD (p-for-trend < 0.0001, 0.0014, and < 0.0001, respectively). The adjusted odds ratios per each 500 g of MBW decrease were 1.167 (95% confidence interval [CI]: 1.118-1.218) for PTD, 1.174 (95% CI: 1.070-1.287) for early PTD and 1.151 (95% CI: 1.098-1.206) for late PTD. The effect size of the association of MBW with early PTD was similar to that with late PTD. This study demonstrated for the first time an association of a low MBW with PTD, early PTD, and late PTD in a Japanese nationwide cohort.

Polycomb Repressive Complex 1.1 Component, BCOR, Promotes Syncytiotrophoblast Differentiation in Mice and Humans.

Early defects in placenta development are thought to underlie a range of adverse pregnancy conditions including miscarriage, fetal growth abnormalities, preeclampsia, and stillbirth. Differentiating trophoblast stem cells undergo a choreographed allocation of syncytiotrophoblast and extravillous trophoblast cells in response to signaling cues from the developing fetus and the uterine environment. The expression and activity of transcription factors and chromatin modifying enzymes change during differentiation to appropriately reshape the chromatin landscape in each cell type. We have previously found in mice that extraembryonic loss of BCOR, a conserved component of the epigenetic silencing complex Polycomb Repressive Complex 1.1 (PRC1.1), leads to a reduced labyrinth and expanded trophoblast giant cell population in the placenta. Molecular analysis of wild-type and BCOR loss-of-function male and female placentas by RNA-seq identified gene expression changes as early as E6.5. We found that BCOR is required to down regulate stem cell genes and repress factors that promote alternate lineages which leads to reduced levels of syncytiotrophoblasts. ChIP-seq experiments identified a number of directly bound functional targets including Pdgfa and Wnt7b . In humans, BCOR is mutated in X-linked syndromes involving fetal growth restriction and females with a heterozygous null mutation in BCOR can experience recurrent miscarriages. To establish a direct role for BCOR in human placental development, we used CRISPR/Cas9 to knockout BCOR in male (CT29) and female (CT30) human trophoblast stem cells. Mutant cell lines retained capacity for induced differentiation into syncytiotrophoblast and extravillous trophoblasts and exhibited minimal changes in gene expression. However, in 3D cell culture using trophoblast organoid media, BCOR knockout lines had significantly altered gene expression including homologs of stem cell genes upregulated in Bcor knockout mice. CUT&RUN experiments in self-renewing and 3D cell culture identified genes directly bound by BCOR. Single cell profiling of wild type, knockout, and a P85L pathogenic knock-in BCOR mutation showed a reduced capacity to differentiate into syncytiotrophoblasts after four days of differentiation. Together, these results suggest that BCOR is a conserved regulator of trophoblast development that represses stem cell genes during differentiation and maintains lineage fidelity by repressing genes that promote alternate cell fates.

Modeling embryo-endometrial interface recapitulating human embryo implantation.

The initiation of human pregnancy is marked by the implantation of an embryo into the uterine environment; however, the underlying mechanisms remain largely elusive. To address this knowledge gap, we developed hormone-responsive endometrial organoids (EMO), termed apical-out (AO)-EMO, which emulate the in vivo architecture of endometrial tissue. The AO-EMO comprise an exposed apical epithelium surface, dense stromal cells, and a self-formed endothelial network. When cocultured with human embryonic stem cell-derived blastoids, the three-dimensional feto-maternal assembloid system recapitulates critical implantation stages, including apposition, adhesion, and invasion. Endometrial epithelial cells were subsequently disrupted by syncytial cells, which invade and fuse with endometrial stromal cells. We validated this fusion of syncytiotrophoblasts and stromal cells using human blastocysts. Our model provides a foundation for investigating embryo implantation and feto-maternal interactions, offering valuable insights for advancing reproductive medicine.

Maternal birth weight as an indicator of early and late gestational diabetes mellitus: The Japan Environment and Children's Study.

AIMS: This study aimed to investigate the association of maternal birth weight (MBW) with early and late gestational diabetes mellitus (GDM). METHODS: A total of 69318 pregnant Japanese women were included in this birth cohort study. The associations between maternal birth weight and early gestational diabetes mellitus (diagnosed at <24 gestational weeks) and late GDM (diagnosed at ≥24 gestational weeks) were investigated using a multinomial logistic regression model, with an maternal birth weight of 3000-3499 g as the reference category. RESULTS: Lower maternal birth weight was associated with higher odds of developing early and late gestational diabetes mellitus (P < 0.0001 and P < 0.0001, respectively). The adjusted odds ratios (aORs) for early gestational diabetes mellitus in participants with a MBW of <2500 g and 2500-2999 g were 1.345 (95% confidence interval [CI]: 0.912-1.984) and 1.338 (95% CI: 1.098-1.629), respectively. The aORs for late gestational diabetes mellitus in participants with a MBW of <2500 g and 2500-2999 g were, 1.657 (95% CI: 1.298-2.115) and 1.218 (95% CI: 1.058-1.402), respectively. CONCLUSIONS: Regardless of the gestational age when gestational diabetes mellitus was diagnosed, a lower maternal birth weight was associated with an increased risk of gestational diabetes mellitus. Furthermore, the association of a MBW <2500 g with late gestational diabetes mellitus tended to be stronger than that with early gestational diabetes mellitus.

Association between Maternal Birth Weight and Prevalence of Congenital Malformations in Offspring: The Japanese Environment and Children's Study.

Congenital malformations are functional and structural alterations in embryonic or foetal development resulting from a variety of factors including maternal health status. This study aimed to investigate the association between maternal birth weight (MBW) and the prevalence of congenital malformations in offspring using data from a nationwide birth cohort study in Japan including 103,060 pregnancies. A binary logistic regression model with adjustment for various covariates revealed that an MBW of <2500 g (low MBW) was associated with an increased risk of congenital heart disease (adjusted odds ratio: 1.388, [95% confidence interval: 1.075-1.792]), angioma (1.491 [1.079-2.059]), and inguinal hernia (1.746, [1.189-2.565]), while those with an MBW of ≥4000 g (high MBW) were associated with congenital anomalies of the urinary tract (2.194, [1.261-3.819]) and arrhythmia (1.775, [1.157-2.725]) compared with those with an MBW of 3000-3499 g. Low MBW was associated with cleft lip and/or palate (1.473, [1.052-2.064]), congenital heart disease (1.615, [1.119-2.332]), genital organs (1.648, [1.130-2.405]), hypospadias (1.804, [1.130-2.881]), and inguinal hernia (1.484, [1.189-1.851]) in male infants and CAKUT (1.619, [1.154-2.273]) in female infants, whereas high MBW was associated with congenital heart disease (1.745, [1.058-2.877]) and CAKUT (2.470, [1.350-4.517]) in male infants. The present study is the first to demonstrate a link between MBW and congenital malformations in Japanese children. While these results must be interpreted with caution, MBW should be considered a major predictor of congenital malformation risk.

Trophoblast stem cell-based organoid models of the human placental barrier.

Human placental villi have essential roles in producing hormones, mediating nutrient and waste exchange, and protecting the fetus from exposure to xenobiotics. Human trophoblast organoids that recapitulate the structure of villi could provide an important in vitro tool to understand placental development and the transplacental passage of xenobiotics. However, such organoids do not currently exist. Here we describe the generation of trophoblast organoids using human trophoblast stem (TS) cells. Following treatment with three kinds of culture medium, TS cells form spherical organoids with a single outer layer of syncytiotrophoblast (ST) cells that display a barrier function. Furthermore, we develop a column-type ST barrier model based on the culture condition of the trophoblast organoids. The bottom membrane of the column is almost entirely covered with syndecan 1-positive ST cells. The barrier integrity and maturation levels of the model are confirmed by measuring transepithelial/transendothelial electrical resistance (TEER) and the amount of human chorionic gonadotropin. Further analysis reveals that the model can be used to derive the apparent permeability coefficients of model compounds. In addition to providing a suite of tools for the study of placental development, our trophoblast models allow the evaluation of compound transfer and toxicity, which will facilitate drug development.

NOTUM-MEDIATED WNT SILENCING DRIVES EXTRAVILLOUS TROPHOBLAST CELL LINEAGE DEVELOPMENT.

Trophoblast stem (TS) cells have the unique capacity to differentiate into specialized cell types, including extravillous trophoblast (EVT) cells. EVT cells invade into and transform the uterus where they act to remodel the vasculature facilitating the redirection of maternal nutrients to the developing fetus. Disruptions in EVT cell development and function are at the core of pregnancy-related disease. WNT-activated signal transduction is a conserved regulator of morphogenesis of many organ systems, including the placenta. In human TS cells, activation of canonical WNT signaling is critical for maintenance of the TS cell stem state and its downregulation accompanies EVT cell differentiation. We show that aberrant WNT signaling undermines EVT cell differentiation. Notum, palmitoleoyl-protein carboxylesterase (NOTUM), a negative regulator of canonical WNT signaling, was prominently expressed in first trimester EVT cells developing in situ and upregulated in EVT cells derived from human TS cells. Furthermore, NOTUM was required for optimal human TS cell differentiation to EVT cells. Activation of NOTUM in EVT cells is driven, at least in part, by endothelial PAS domain 1 (also called hypoxia-inducible factor 2 alpha). Collectively, our findings indicate that canonical WNT signaling is essential for maintenance of human trophoblast cell stemness and regulation of human TS cell differentiation. Downregulation of canonical WNT signaling via the actions of NOTUM is required for optimal EVT cell differentiation.

Long-Term Outcomes of Simple Endovascular Aneurysm Repair Based on the Initial Aortic Diameter.

PURPOSE: We aimed to investigate the effects of initial abdominal aortic aneurysm (AAA) diameter on aneurysmal sac expansion/shrinkage, endoleaks, and reintervention postelective simple endovascular aneurysm repair (EVAR). METHODS: Overall, 228 patients monitored for >1 year after EVAR were analyzed. Male and female participants with initial AAA diameters <55 mm and <50 mm, respectively, composed the small group (group S), while those with initial AAA diameters ≥55 mm (men) and ≥50 mm (women) composed the large group (group L). Aneurysmal sac expansion of 10 mm and/or reintervention during follow-up (composite event) and its related factors were evaluated. RESULTS: The 5-year freedom from composite event rate was significantly higher in group S (92.4 ± 2.8%) than that in group L (79.1 ± 4.9%; P <0.01). Multivariate analysis revealed AAA diameters before EVAR in group S (hazard ratio, 0.38; 95% confidence interval, 0.18-0.81; P = 0.01) and type II endoleak (T2EL) at discharge (hazard ratio, 2.83; 95% confidence interval, 1.29-6.20; P <0.01) as factors associated with the composite event. The freedom from composite event rate decreased to 51 ± 13% at 5 years in group L with T2EL. CONCLUSIONS: Group S had high freedom from composite event rate; in group L, the rate decreased to 51% at 5 years with T2EL at discharge.

Cartilage tissues regulate systemic aging via ectonucleotide pyrophosphatase/phosphodiesterase 1 in mice.

Aging presents fundamental health concerns worldwide; however, mechanisms underlying how aging is regulated are not fully understood. Here, we show that cartilage regulates aging by controlling phosphate metabolism via ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1). We newly established an Enpp1 reporter mouse, in which an EGFP-luciferase sequence was knocked-in at the Enpp1 gene start codon (Enpp1/EGFP-luciferase), enabling detection of Enpp1 expression in cartilage tissues of resultant mice. We then established a cartilage-specific Enpp1 conditional knockout mouse (Enpp1 cKO) by generating Enpp1 flox mice and crossing them with cartilage-specific type 2 collagen Cre mice. Relative to WT controls, Enpp1 cKO mice exhibited phenotypes resembling human aging, such as short life span, ectopic calcifications, and osteoporosis, as well as significantly lower serum pyrophosphate levels. We also observed significant weight loss and worsening of osteoporosis in Enpp1 cKO mice under phosphate overload conditions, similar to global Enpp1-deficient mice. Aging phenotypes seen in Enpp1 cKO mice under phosphate overload conditions were rescued by a low vitamin D diet, even under high phosphate conditions. These findings suggest overall that cartilage tissue plays an important role in regulating systemic aging via Enpp1.

CRISPR screening in human trophoblast stem cells reveals both shared and distinct aspects of human and mouse placental development.

The placenta serves as the interface between the mother and fetus, facilitating the exchange of gases and nutrients between their separate blood circulation systems. Trophoblasts in the placenta play a central role in this process. Our current understanding of mammalian trophoblast development relies largely on mouse models. However, given the diversification of mammalian placentas, findings from the mouse placenta cannot be readily extrapolated to other mammalian species, including humans. To fill this knowledge gap, we performed CRISPR knockout screening in human trophoblast stem cells (hTSCs). We targeted genes essential for mouse placental development and identified more than 100 genes as critical regulators in both human hTSCs and mouse placentas. Among them, we further characterized in detail two transcription factors, DLX3 and GCM1, and revealed their essential roles in hTSC differentiation. Moreover, a gene function-based comparison between human and mouse trophoblast subtypes suggests that their relationship may differ significantly from previous assumptions based on tissue localization or cellular function. Notably, our data reveal that hTSCs may not be analogous to mouse TSCs or the extraembryonic ectoderm (ExE) in which in vivo TSCs reside. Instead, hTSCs may be analogous to progenitor cells in the mouse ectoplacental cone and chorion. This finding is consistent with the absence of ExE-like structures during human placental development. Our data not only deepen our understanding of human trophoblast development but also facilitate cross-species comparison of mammalian placentas.

The IL-17-IL-17RA axis is required to promote osteosarcoma progression in mice.

Osteosarcoma is rare but is the most common bone tumor. Diagnostic tools such as magnetic resonance imaging development of chemotherapeutic agents have increased the survival rate in osteosarcoma patients, although 5-year survival has plateaued at 70%. Thus, development of new treatment approaches is needed. Here, we report that IL-17, a proinflammatory cytokine, increases osteosarcoma mortality in a mouse model with AX osteosarcoma cells. AX cell transplantation into wild-type mice resulted in 100% mortality due to ectopic ossification and multi-organ metastasis. However, AX cell transplantation into IL-17-deficient mice significantly prolonged survival relative to controls. CD4-positive cells adjacent to osteosarcoma cells express IL-17, while osteosarcoma cells express the IL-17 receptor IL-17RA. Although AX cells can undergo osteoblast differentiation, as can patient osteosarcoma cells, IL-17 significantly inhibited that differentiation, indicating that IL-17 maintains AX cells in the undifferentiated state seen in malignant tumors. By contrast, IL-17RA-deficient mice transplanted with AX cells showed survival comparable to wild-type mice transplanted with AX cells. Biopsy specimens collected from osteosarcoma patients showed higher expression of IL-17RA compared to IL-17. These findings suggest that IL-17 is essential to maintain osteosarcoma cells in an undifferentiated state and could be a therapeutic target for suppressing tumorigenesis.

Timing of Primary Tooth Eruption in Infants Observed by Their Parents.

BACKGROUND: The timing of primary teeth eruption is a visible indicator of infant physical growth other than body weight or height. It also reflects neurological integrity and development as well as nutrition, socioeconomic state, or underlying diseases. Therefore, the timing of primary teeth eruption is one of the major concerns for parents in health checkups for infants and children. However, the detailed developmental timing of teeth eruption differs depending on the survey methodology, country, or generation. We hypothesized that the timing of primary teeth eruption differs between the medical checkup by dentists and the daily records by parents. METHODS: We conducted a questionnaire survey on the date of eruption of primary teeth as an adjunct study among Miyagi Regional Center participants in the Japan Environment and Children's Study (JECS), a large-scale birth cohort study. A total of 1695 responses (3793 participants) were analyzed. RESULTS: The median ages of eruption were 7.1 months (male) and 7.6 months (female) for mandibular primary central incisors, 8.7 months (male) and 9.2 months (female) for maxillary primary central incisors, 10.0 months (male) and 10.3 months (female) for maxillary primary lateral incisors, and 10.4 months (male) and 10.8 months (female) for mandibular primary lateral incisors, which were earlier than the reported timings based on dental check-ups. Comparing the eruption time of preterm and term infants, the eruption time was earlier in preterm infants in the corrected ages. CONCLUSIONS: The eruption timing observed and described by the parents is earlier than that examined by dentists at regular check-ups. In addition to examining the primary teeth eruption of full-term birth children, we also examined that of preterm birth children because of the increasing number of premature births. To the best of our knowledge, this is the first report from a large cohort study to clarify the eruption time of primary teeth monitored by parents.

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.