Bell Stage Research Articles

Association of hypertensive disorder of pregnancy with necrotizing enterocolitis in very preterm infants: A retrospective cohort study.

Hypertensive disorders of pregnancy (HDP) may affect fetal development and result in preterm delivery. Necrotizing enterocolitis (NEC) is a severe gastrointestinal emergency in very preterm infants (VPIs, gestational age less than 32 weeks). The relationship between maternal HDP and NEC is controversial. Objective To investigate the association between maternal HDP and NEC in VPIs.This was a multicenter retrospective cohort study based on the data from the Chinese Neonatal Network (CHNN) which were collected between January 1, 2019 and December 31, 2021. Preterm infants born between 24+0 and 31+6 weeks of gestation were divided into HDP and no-HDP groups according to the 2015 Chinese guidelines for HDP. The primary outcome was the incidence of Bell's stage II or higher NEC. Secondary outcomes included mortality and spontaneous intestinal perforation (SIP). Of 27,660 women were included in the study analysis, 5405 (19.5%) were HDP and 22256 (80.5%) were no-HDP. NEC occurred in 5.2% (283/5,404) among HDP mothers and 5.3% (1,191/22,256) among no-HDP mothers. No significant association was observed between HDP and Bell's stage II or higher NEC (aOR 0.87, 95% CI [0.72, 1.05]). However, even after adjustment, maternal HDP appeared to be protective for NEC requiring surgical intervention (aOR 0.60, 95% CI [0.43, 0.83]). There was no significant correlation between maternal HDP and neonatal mortality and SIP. Maternal HDP was not significantly associated with the incidence of Bell's stage II or higher NEC. However, it was associated with the lower rate of NEC requiring surgical intervention.

005. The Role of Interleukin-8 in Assisting the Diagnosis of Necrotizing Enterocolitis (NEC) Bell’s Stage I and II in Premature Infants: Literature Review

Background: Necrotizing enterocolitis (NEC) is one of the leading causes of morbidity and mortality in infants, commonly associated with extremely low birth weight (ELBW), low birth weight (LBW), and prematurity. The clinical symptoms are nonspecific and various. This poses a challenge in the early diagnosis. The incidence worldwide ranges from 0.3 to 2.4 per 1,000 live births, with mortality rates reaching 50%. This study aims to assess the role of biomarker IL-8 in diagnosing NEC Bell's stage I and II. Methods: This study is a literature review, conducted electronically in databases including Cochrane Library, PubMed, ScienceDirect, and EBSCO using the keywords: ("Interleukin-8" OR "IL-8") AND ("Necrotizing enterocolitis" OR "NEC" OR “Early NEC” OR “Stage I-II NEC”). Inclusion criteria are study in human, English fulltext within 5 years. Exclusion criteria is comparing NEC any stage with control patients, Results: Based on a review of 23 literatures, there were 6 studies met the inclusion criteria for analysis. IL-8 shows varying sensitivity and specificity as a serum biomarker for diagnosing NEC. Statistical evaluations in previous studies emphasized that significantly elevated levels of biomarker IL-8 were predominantly found in the serum of premature infants with NEC, indicating it could be a potential biomarker for diagnosis with NEC Bell's stage I and II. Conclusion: The measurement of biomarker IL-8 in blood has not yet been established as a reliable diagnostic modality for NEC Bell's stage I and II, indicating the need for further investigation.

Conservative Management of Necrotizing Enterocolitis in Newborns: Incidence and Management of Intestinal Strictures.

Necrotizing enterocolitis (NEC) is one of the main causes of acute abdomen in neonates. Surgical treatment entails important morbidity and mortality and conservative management, when possible, offers better outcomes. Post-NEC intestinal strictures are one of the main complications. Retrospective analysis from June 2011 to November 2022 of post-NEC strictures (PNS) after conservative management of neonates diagnosed with NEC (modified Bell stage IIA or higher) at a tertiary neonatal surgery center. Out of 219 NEC, 126 received initial conservative management (57.5%), 24 (19%) of which eventually underwent surgery for PNS. Average gestational age and weight at birth of our cohort were 31.3 ± 4.9 weeks and 1,694 ± 1,009 g.PNS diagnosis was made 38.4 ± 16.5 days after the NEC episode. 6/24 (25%) were asymptomatic and diagnosed by screening enema, 11 (46%) presented signs of intestinal obstruction before the enema could be performed and 7 (29%) after a normal previous protocol study.Median age at PNS surgery was 56 ± 17.9 days. A total of 2/3 strictures were found in cecum, ascendent, and transverse colon. Primary resection and anastomosis were performed in all cases. Feeds were restarted on postoperative day 4.3 ± 2.9. Two cases presented anastomotic complications (1 dehiscence and 1 stenosis), and no deaths were recorded. PNS is a frequent complication after conservative management. Deffered surgical treatment after the acute NEC episode is resolved allows for safer surgeries (since patients have reached hemodynamical stability and overcome septic shock), shorter resections, and favorable postoperative outcomes.

Organotypic 3D cellular models mimicking the epithelio-ectomesenchymal bi-layer during odontogenesis.

Odontogenesis, the intricate process of tooth development, involves complex interactions between oral ectoderm epithelial cells and ectomesenchymal cells derived from the cephalic neural crest, regulated by major signaling pathways. Dental developmental anomalies provide valuable insights for clinical diagnosis of rare diseases. More than 30% of rare diseases patients who undergo molecular analysis suffer from diagnostic errancy. In the search for up-to-date technologies and methods to study the pathophysiology of new candidate genetic variants, causing tooth mineralized tissues anomalies, we have developed an original model of tooth organoids with human or mouse cell lines of ameloblast-like cells and odontoblasts derived from the pulp. This in vitro 3D cellular model reproducing the two main compartments of the bell stage of tooth development between ameloblasts and odontoblasts, specific to enamel and dentin morphogenesis, respectively, mimics the epithelio-mesenchymal interactions during the dental bell stage of tooth morphogenesis and will facilitate the study of enamel and dentin genetic anomalies, allowing the functional validation of newly identified mutations (variants of uncertain significance -VUS- or new candidate genes).

Single-cell RNA-seq analysis of rat molars reveals cell identity and driver genes associated with dental mesenchymal cell differentiation

BackgroundThe molecular mechanisms and signaling pathways involved in tooth morphogenesis have been the research focus in the fields of tooth and bone development. However, the cell population in molars at the late bell stage and the mechanisms of hard tissue formation and mineralization remain limited knowledge.ResultsHere, we used the rat mandibular first and second molars as models to perform single-cell RNA sequencing (scRNA-seq) analysis to investigate cell identity and driver genes related to dental mesenchymal cell differentiation during the late bell hard tissue formation stage. We identified seven main cell types and investigated the heterogeneity of mesenchymal cells. Subsequently, we identified novel cell marker genes, including Pclo in dental follicle cells, Wnt10a in pre-odontoblasts, Fst and Igfbp2 in periodontal ligament cells, and validated the expression of Igfbp3 in the apical pulp. The dynamic model revealed three differentiation trajectories within mesenchymal cells, originating from two types of dental follicle cells and apical pulp cells. Apical pulp cell differentiation is associated with the genes Ptn and Satb2, while dental follicle cell differentiation is associated with the genes Tnc, Vim, Slc26a7, and Fgfr1. Cluster-specific regulons were analyzed by pySCENIC. In addition, the odontogenic function of driver gene TNC was verified in the odontoblastic differentiation of human dental pulp stem cells. The expression of osteoclast differentiation factors was found to be increased in macrophages of the mandibular first molar.ConclusionsOur results revealed the cell heterogeneity of molars in the late bell stage and identified driver genes associated with dental mesenchymal cell differentiation. These findings provide potential targets for diagnosing dental hard tissue diseases and tooth regeneration.

Rapidly progressive necrotizing enterocolitis: Risk factors and a predictive model.

Rapidly progressive necrotizing enterocolitis (RP-NEC) is a particular subtype of NEC known for its rapid progression and high mortality rate. The objective of this study was to establish a predictive model for RP-NEC. This was a retrospective single-center cohort study. Patients were newborn infants with NEC (Bell's stage ≥ IIB) admitted from January 1, 2016 to December 31, 2023. The primary outcome was RP-NEC defined as the need for surgical intervention and/or death within 48 hours of the onset of NEC. Totally 334 newborn infants were included, among which 82 (24.6%) were RP-NEC cases with a gestation age 34.1 (31.0, 37.0) weeks and birth weight 2100 (1413, 2800) g. Plasma sodium <135 mmol/L, C-reactive protein ≥10 mg/L, platelet count <100 × 109/L, lymphocyte count <1.5 × 109/L, pH <7.2 in blood gas, and ascites at NEC onset were identified as independent risk factors for RP-NEC. The model established presented an AUC value of 0.983 (95% CI 0.97-0.99). The calibration curve for validation was applied revealing a slope close to unity while the Hosmer-Lemeshow test yielded χ2 = 2.550 (p = 0.636). The predictive model established on the above 6 items of RP-NEC is highly promising. Currently, there is a paucity of research on this specific type of severe necrotizing enterocolitis (NEC) characterized by rapid progression. Our study was to investigate the risk factors associated with surgical intervention and/or death within 48 hours following onset in infants with NEC, establish a predictive model for infants with rapidly progressive NEC. The new data presented in this study was the ROC curve combining the above factors as well as hyponatremia.

Impact of postoperative necrotizing enterocolitis after neonatal cardiac surgery on neurodevelopmental outcome at 1 year of age.

We analyzed the impact of postoperative necrotizing enterocolitis (NEC) after cardiac surgery in neonatal age on neurodevelopmental (ND) outcome at 1 year of age. Using data from the Swiss Neurodevelopmental Outcome Registry for Children with Congenital Heart Disease (ORCHID), we analyzed perioperative variables including postoperative NEC (Bell's stage ≥2) and 1-year ND outcome (Bayley III). The included patients (n = 101) had congenital heart disease (CHD), categorized as follows: 77 underwent biventricular repair for CHD with two functional chambers, 22 underwent staged palliation until the Fontan procedure for CHD with single ventricle physiology (n = 22), or 4 underwent single ventricle palliation or biventricular repair for borderline CHD (n = 4). Neonatal cardiopulmonary bypass (CBP) surgery was performed at a median age (IQR) of 8 (6) days. NEC occurred in 16 patients. Intensive care unit (ICU) length of stay (LOS) and the total duration of the hospitalization were longer in children with NEC than those in others (14 with vs. 8 days without NEC, p < 0.05; 49 with vs. 32 days without NEC, p < 0.05). The Bayley III scores of the analyzed patients determined at an age of 11.5 ± 1.5 months showed cognitive (CCS) (102.2 ± 15.0) and language scores (LCS) (93.8 ± 13.1) in the normal range and motor composite scores (MCS) (88.7 ± 15.9) in the low-normal range. After adjusting for socioeconomic status and CHD type, patients with NEC had lower CCS scores [β = -11.2 (SE 5.6), p = 0.049]. Using a cumulative risk score including NEC, we found a higher risk score to be associated with both lower CCS [β = -2.8 (SE 1.3), p = 0.030] and lower MCS [β = -3.20 (SE 1.3), p = 0.016]. Postoperative NEC is associated with longer ICU and hospital LOS and contributes together with other complications to impaired ND outcome at 1 year of age. In the future, national and international patient registries may provide the opportunity to analyze large cohorts and better identify the impact of modifiable perioperative risk factors on ND outcome. ClinicalTrials.gov identifier: NCT05996211.

Breast Milk and Necrotizing Enterocolitis in Congenital Heart Disease: A Case-Control Study.

Background: Necrotizing enterocolitis (NEC) is a complication that can affect infants with congenital heart disease (CHD). The objective of this study is to determine whether breast milk, which is associated with decreased incidence of NEC in preterm infants, is protective in infants with CHD. Methods: Retrospective case-control study of infants ≥ 33 weeks gestational age with CHD who underwent cardiac surgery during their admission to the Infant Cardiac Unit from 2008 to 2017. Cases were defined as infants with modified Bell's stage ≥ II NEC. Controls were matched by date of birth, gestational age, and pre- or postcardiac surgery feed initiation. Results: A total of 926 infants with gestational age ≥ 33 weeks and CHD were admitted; 18 cases of NEC were identified and compared with 84 controls. Breast milk intake was higher in controls, but this difference was not statistically significant. Single ventricle (SV) physiology was identified as an independent risk factor for NEC by multivariable analysis. Analysis of infants with SV physiology demonstrated that median age at time of surgery was 9 days (interquartile range [IQR], 7-12) in NEC cases and 5 days (IQR, 4-9) in controls (P = .02). Conclusions: While this study is inconclusive with regard to feeding composition and risk of NEC in infants with CHD, the trend toward greater intake of breast milk in the control group suggests that breast milk may be protective for these infants. Infants with SV physiology are at high risk for NEC. Earlier time to stage I palliation may be a modifiable risk factor for NEC.

Spatial and temporal gene expression patterns during early human odontogenesis process.

Studies on odontogenesis are of great importance to treat dental abnormalities and tooth loss. However, the odontogenesis process was poorly studied in humans, especially at the early developmental stages. Here, we combined RNA sequencing (RNA-seq) with Laser-capture microdissection (LCM) to establish a spatiotemporal transcriptomic investigation for human deciduous tooth germs at the crucial developmental stage to offer new perspectives to understand tooth development and instruct tooth regeneration. Several hallmark events, including angiogenesis, ossification, axonogenesis, and extracellular matrix (ECM) organization, were identified during odontogenesis in human dental epithelium and mesenchyme from the cap stage to the early bell stage. ECM played an essential role in the shift of tooth-inductive capability. Species comparisons demonstrated these hallmark events both in humans and mice. This study reveals the hallmark events during odontogenesis, enriching the transcriptomic research on human tooth development at the early stage.

Ectopic Activation of Fgf8 in Dental Mesenchyme Causes Incisor Agenesis and Molar Microdontia.

Putatively, tooth agenesis was attributed to the initiation failure of tooth germs, though little is known about the histological and molecular alterations. To address if constitutively active FGF signaling is associated with tooth agenesis, we activated Fgf8 in dental mesenchyme with Osr-cre knock-in allele in mice (Osr2-creKI; Rosa26R-Fgf8) and found incisor agenesis and molar microdontia. The cell survival assay showed tremendous apoptosis in both the Osr2-creKI; Rosa26R-Fgf8 incisor epithelium and mesenchyme, which initiated incisor regression from cap stage. In situ hybridization displayed vanished Shh transcription, and immunostaining exhibited reduced Runx2 expression and enlarged mesenchymal Lef1 domain in Osr2-creKI; Rosa26R-Fgf8 incisors, both of which were suggested to enhance apoptosis. In contrast, Osr2-creKI; Rosa26R-Fgf8 molar germs displayed mildly suppressed Shh transcription, and the increased expression of Ectodin, Runx2 and Lef1. Although mildly smaller than WT controls prenatally, the Osr2-creKI; Rosa26R-Fgf8 molar germs produced a miniature tooth with impaired mineralization after a 6-week sub-renal culture. Intriguingly, the implanted Osr2-creKI; Rosa26R-Fgf8 molar germs exhibited delayed odontoblast differentiation and accelerated ameloblast maturation. Collectively, the ectopically activated Fgf8 in dental mesenchyme caused incisor agenesis by triggering incisor regression and postnatal molar microdontia. Our findings reported tooth agenesis resulting from the regression from the early bell stage and implicated a correlation between tooth agenesis and microdontia.

Umbilical arterial catheter duration as risk factor for Bell's Stage III necrotizing enterocolitis in preterm neonates.

To explore risk factors for Stage-III necrotizing enterocolitis (NEC-III) in preterm neonates. This was a retrospective case-control study of neonates born <33 weeks gestational age (GA) who were admitted to a tertiary neonatal intensive care unit, between 2015 and 2018. NEC-III cases were compared with Stage-II NEC (NEC-II) and non-NEC controls. Two to four non-NEC controls were matched by GA ± 1 week and date of birth ± 3 months, to one NEC-III case. Univariate and multivariate analyses were used to examine risk factors for NEC-III. Of 1360 neonates born <33 weeks, 71 (5.2%) had NEC-II and above, with 46% being NEC-III. Mean age of onset of NEC-III was 13.7 days versus 23.9 days for NEC-II (p = 0.01). Neonates with NEC-III were of lower GA (NEC-III 25.4 weeks, NEC-II 27.3 weeks, and non-NEC 26 weeks; p = 0.0008) and had higher Score for Neonatal Acute Physiology Perinatal Extension-II scores (NEC-III 47.5, NEC-II 28.4 and non-NEC 37, p = 0.003). Multivariate analysis showed duration of umbilical arterial catheter (UAC) >5 days was significantly associated with the development of NEC-III with adjusted odds ratio (AOR) 3.8; 95% confidence interval (CI) (1.05-13.66) for NEC-III versus non-NEC and AOR 5.57; 95% CI (1.65-18.73), p = 0.006 for NEC-III versus NEC-II. Rupture of membranes (ROM) >1 week was associated with NEC-III (AOR 6.93; 95% CI [1.56-30.69] vs. non-NEC and AOR 11.74; 95% CI [1.14-120.34] vs. NEC-II). The increased association of NEC-III with duration of UAC and ROM could be further examined in prospective studies, and an upper limit for UAC duration could be considered in NEC prevention bundles.

Interplay of RUNX2 and KLF4 in initial commitment of odontoblast differentiation.

Odontoblast differentiation is a key process in dentin formation. Mouse dental papilla cells (mDPCs) are pivotal in dentinogenesis through their differentiation into odontoblasts. Odontoblast differentiation is intricately controlled by transcription factors (TFs) in a spatiotemporal manner. Previous research explored the role of RUNX2 and KLF4 in odontoblast lineage commitment, respectively. Building on bioinformatics analysis of our previous ATAC-seq profiling, we hypothesized that KLF4 potentially collaborates with RUNX2 to exert its biological role. To investigate the synergistic effect of multiple TFs in odontoblastic differentiation, we first examined the spatiotemporal expression patterns of RUNX2 and KLF4 in dental papilla at the bell stage using immunostaining techniques. Notably, RUNX2 and KLF4 demonstrated colocalization in preodontoblast. Further, immunoprecipitation and proximity ligation assays verified the interaction between RUNX2 and KLF4 in vitro. Specifically, the C-terminus of RUNX2 was identified as the interacting domain with KLF4. Functional implications of this interaction were investigated using small hairpin RNA-mediated knockdown of Runx2, Klf4, or both. Western blot analysis revealed a marked decrease in DSPP expression, an odontoblast differentiation marker, particularly in the double knockdown condition. Additionally, alizarin red S staining indicated significantly reduced mineralized nodule formation in this group. Collectively, our findings highlight the synergistic interaction between RUNX2 and KLF4 in promoting odontoblast differentiation from mDPCs. This study contributes to a more comprehensive understanding of the regulatory network of TFs governing odontoblast differentiation.

Advancing necrotizing enterocolitis prediction through iterative monitoring.

Necrotizing enterocolitis (NEC) is a severe inflammatory intestinal disease in preterm infants, marked by heightened morbidity and mortality. Timely prediction of NEC is significant in the management of critical neonates. However, it is difficult to predict NEC accurately because of the multi-factorial pathogenesis. This study aimed to develop a prediction model through repeated measurement data to further improve the accuracy of prediction in NEC. We retrospectively collected clinical data of premature infants admitted to the Neonatology Department of the First Affiliated Hospital of Anhui Medical University from January 2016 to December 2023. The infants were categorized into the NEC group (Bell's stage ≥ II) (n=150) and the non-NEC group (n=150). The clinical baseline data of the NEC and non-NEC groups were matched. Laboratory examination indicators were collected on the 1st day, the 7th day after birth, and the day of NEC onset. Univariate and multivariate logistic regression analyses were conducted to identify independent factors influencing NEC. A nomogram was constructed based on these factors to predict NEC. The concordance index and calibration plot were used to assess the efficiency of the nomogram in the training and validation cohorts. This study demonstrated that antenatal steroids, antenatal antibiotics, probiotics treatment before NEC, anion gap (AG, day 7), and mean corpuscular volume (MCV, day 7) were independent risk factors which combined to accurately predict NEC. A nomogram of NEC was created utilizing these five predictors. With an area under the receiver operator characteristic (ROC) curve of 0.835 [95% confidence interval (CI): 0.785-0.884]. Concordance index for the training and validation groups were 0.835 and 0.848, respectively. As the calibration plots indicate, the predicted probability of NEC is highly consistent with the actual observation. The risk estimation nomogram for NEC offers clinical value by guiding early prediction, targeted prevention, and early intervention strategies for NEC.

Necrotizing enterocolitis: a potential protective role for intestinal alkaline phosphatase as lipopolysaccharide detoxifying enzyme.

Necrotizing enterocolitis (NEC) is a life-threatening inflammatory disease. Its onset might be triggered by Toll-Like Receptor 4 (TLR4) activation via bacterial lipopolysaccharide (LPS). We hypothesize that a deficiency of intestinal alkaline phosphatase (IAP), an enzyme secreted by enterocytes that dephosphorylates LPS, may contribute to NEC development. In this prospective pilot study, we analyzed intestinal resection specimens from surgical NEC patients, and from patients undergoing Roux-Y reconstruction for hepatobiliary disease as controls. We assessed IAP activity via enzymatic stainings and assays and explored IAP and TLR4 co-localization through immunofluorescence. The study population consisted of five NEC patients (two Bell's stage IIb and three-stage IIIb, median (IQR) gestational age 25 (24-28) weeks, postmenstrual age at diagnosis 28 (26-31) weeks) and 11 controls (unknown age). There was significantly lower IAP staining in NEC resection specimens [49 (41-50) U/g of protein] compared to controls [115 (76-144), P = 0.03]. LPS-dephosphorylating activity was also lower in NEC patients [0.06 (0-0.1)] than in controls [0.3 (0.2-0.5), P = 0.003]. Furthermore, we observed colocalization of IAP and TLR4 in NEC resection specimens. This study suggests a significantly lower IAP level in resection specimens of NEC patients compared to controls. This lower IAP activity suggests a potential role of IAP as a protective agent in the gut, which needs further confirmation in larger cohorts.

Prenatal and postnatal development of New Zealand white rabbit (Oryctolagus cuniculus) teeth: histological and computed tomography aspects

The study reveals a lack of histomorphogenesis in New Zealand white rabbit teeth. The teeth development was examined through sequential histological segments in 24 rabbits from prenatal ages (E19, E21, E23, E25, and E28), neonates (E30), and postnatal age (1 week and 2 weeks); (three animal specimens at each age stage). Rabbit teeth first appeared at 19 days of prenatal life (E19) as an ectodermal epithelial thickening on each side of the mouth opening. At E21, the bud of upper incisor tooth appeared as an epithelial bud, which composed of many condensed epithelium cells, was simply identified from the larger with less condensed vestibular lamina, and was surrounded by mesenchymal connective tissue while the lower incisor took the cap stage. At (E23), tooth regular construction is formed from enamel, dentine, and pulp cavity. Peg incisor appearance (supplementary and assistant incisors) is visible at the lingual surface of the upper major incisor. Teeth prenatal development went through successive stages like initiation, bud, cap, late bell, maturation, and crown stages. The first initiation phase of tooth formation was seen as ectodermal epithelial cell collection at (E19). Bud stage saw on upper incisor tooth, while in cap structure in lower incisor teeth at (E19). A cap-formed tooth is composed of the enamel organ and fundamentally dense mesenchymal tissue. Enamel organs are segmented into three distinct layers: the external tooth enamel epithelial, the internal tooth enamel epithelial, and finally the stellate reticular layer. The cement layer covered teeth all around on enamel on both the labial and lingual sides while not contacting the dentine on the lateral side, forming enamel space. Teeth develop consistently all through life; they have expanded enamel thickness; they are diphyodont teeth; they have two continuous dentitions; they are deciduous and perpetual, with long crown teeth and an open root.

The temporal protein signature analyses of developing human deciduous molar tooth germ.

The tooth serves as an exemplary model for developmental studies, encompassing epithelial-mesenchymal transition and cell differentiation. The essential factors and pathways identified in tooth development will help understand the natural development process and the malformations of mineralized tissues such as skeleton. The time-dependent proteomic changes were investigated through the proteomics of healthy human molars during embryonic stages, ranging from the cap-to-early bell stage. A comprehensive analysis revealed 713 differentially expressed proteins (DEPs) exhibiting five distinct temporal expression patterns. Through the application of weighted gene co-expression network analysis (WGCNA), 24 potential driver proteins of tooth development were screened, including CHID1, RAP1GDS1, HAPLN3, AKAP12, WLS, GSS, DDAH1, CLSTN1, AFM, RBP1, AGO1, SET, HMGB2, HMGB1, ANP32A, SPON1, FREM1, C8B, PRPS2, FCHO2, PPP1R12A, GPALPP1, U2AF2, and RCC2. Then, the proteomics and transcriptomics expression patterns of these proteins were further compared, complemented by single-cell RNA-sequencing (scRNA-seq). In summary, this study not only offers a wealth of information regarding the molecular intricacies of human embryonic epithelial and mesenchymal cell differentiation but also serves as an invaluable resource for future mechanistic inquiries into tooth development.

Biomarkers in the Severity of Necrotizing Enterocolitis in Preterm Infants: A Pilot Study.

The occurrence of necrotizing enterocolitis (NEC) is a common and severe disease of the digestive system in neonates. This study aims to assess the value of the intestinal tissue oxygen saturation (rintSO2) combined with the levels of procalcitonin (PCT) and mean platelet volume (MPV) in predicting the severity of NEC in preterm infants. This experiment was a retrospective cohort study conducted in the Department of Pediatrics, Hongqi Hospital Affiliated to Mudanjiang Medical University between January 2017 and July 2022. Premature neonates with NEC were enrolled and divided into mild-moderate NEC group and severe NEC group according to Bell's stage. The general information data, rintSO2 and blood parameters such as the white blood cell (WBC) count, platelet count (PLT), PCT, MPV, red blood cell distribution width (RDW), hemoglobin (Hb), C-reactive protein (CRP) were compared between the two groups. A total of 122 patients were enrolled, including 79 mild-moderate NEC and 43 severe NEC. The rintSO2 was lower in severe group than in mild-moderate group (P = 0.042), the PCT and MPV were both higher in severe group than in mild-moderate group (P = 0.048, P = 0.049). The results of logistic regression suggested that the rintSO2 (OR = 1.491, P = 0.003), PCT (OR = 3.071, P = 0.001) and MPV (OR = 4.027, P = 0.015) were independent predictive factors for severity of NEC. The area under the curve (AUC) of the rintSO2 combined with PCT and MPV showed good diagnostic ability in the severity of NEC. The rintSO2 combined with PCT and MPV may be considered as the early biomarkers in the severity of NEC and could help us to diagnose the case early with early treatment with better prognosis.

Regulatory role of N-myc downregulated genes in amelogenesis in rats.

Cytodifferentiation of odontogenic cells, a late stage event in odontogenesis is based on gene regulation. However, studies on the identification of the involved genes are scarce. The present study aimed to search for molecules for the cytodifferentiation of ameloblastic cells in rats. Differential display-PCR revealed a differentially expressed gene between cap/early bell stage and hard tissue formation stage in molars. This gene was identified as N-myc Downregulated Gene 1 (Ndrg1), which is the first report in tooth development. Real time PCR and western blotting confirmed that the mRNA level of Ndrg1 was higher during enamel formation than the cap stage. Ndrg1 expression was upregulated in the early bell, crown, and root stages in a time-dependent manner. These patterns of expression were similar in Ndrg2, but Ndrg3 and Ndrg4 levels did not change during the developmental stages. Immunofluorescence revealed that strong immunoreactivity against Ndrg1 were detected in differentiated ameloblasts only, not inner enamel epithelium, odontoblasts and ameloblastic cells in defected enamel regions. Alkaline phosphatase and alizarin red s stains along with real time PCR, revealed that Ndrg1 and Ndrg2 were involved in cytodifferentiation and enamel matrix mineralization by selectively regulating amelogenin and ameloblastin genes in SF2 ameloblastic cells. These results suggest that Ndrg may play a crucial functional role in the cytodifferentiation of ameloblasts for amelogenesis.

Morphogenesis of fungiform papillae in developing miniature pigs

ObjectiveFungiform papillae contain taste buds and play a critical role in mastication and the gustatory system. In this study, we report a series of sequential observations of organogenesis of fungiform papillae in miniature pigs, as well as changes in the expression of BMP2, BMP4, Wnt5a, Sox2, and Notch1 signaling pathway components. DesignIn this study, we investigated the spatiotemporal expression patterns of BMP, Wnt, Sox2 and Notch in the fungiform papillae of miniature pigs at the bud stage (E40), cap stage (E50) and bell stage (E60). Pregnant miniature pigs were obtained, and the samples were processed for histological staining. Immunohistochemistry and real-time PCR were used to detect the mRNA and protein expression levels of BMP2, BMP4, Wnt5a, Sox2, and Notch1. ResultsAt E40, fungiform papillae were present on the anterior two-thirds of the tongue in a specific array and pattern. The fungiform papillae were enlarged and basically developed at E50 and were largest at the earlier stage (E60). Most of the BMP2 was concentrated in the epithelial layer and the connective tissue core of the fungal papilloma and gradually accumulated from E40-E60. BMP-4 was weakly expressed in the fungiform papillae epithelia, but BMP-4-positive cells were also observed in the developing tongue muscle at E50 and E60. Wnt5a-positive cells were observed in the fungiform papillae epithelia and developing tongue muscle at all three time points. Sox2-positive cells were observed only in fungiform papillae epithelial cells, and Notch1-positive cells could not be detected. ConclusionsThis study provides primary data regarding the morphogenesis and expression of developmental signals in the fungiform papillae of miniature pigs, establishing a foundation for further research in both this model and humans.

MicroRNA-93-5p regulates odontogenic differentiation and dentin formation via KDM6B

BackgroundEpigenetic factors influence the odontogenic differentiation of dental pulp stem cells and play indispensable roles during tooth development. Some microRNAs can epigenetically regulate other epigenetic factors like DNA methyltransferases and histone modification enzymes, functioning as epigenetic-microRNAs. In our previous study, microarray analysis suggested microRNA-93-5p (miR-93-5p) was differentially expressed during the bell stage in human tooth germ. Prediction tools indicated that miR-93-5p may target lysine-specific demethylase 6B (KDM6B). Therefore, we explored the role of miR-93-5p as an epi-miRNA in tooth development and further investigated the underlying mechanisms of miR-93-5p in regulating odontogenic differentiation and dentin formation.MethodsThe expression pattern of miR-93-5p and KDM6B of dental pulp stem cells (DPSCs) was examined during tooth development and odontogenic differentiation. Dual luciferase reporter and ChIP-qPCR assay were used to validate the target and downstream regulatory genes of miR-93-5p in human DPSCs (hDPSCs). Histological analyses and qPCR assays were conducted for investigating the effects of miR-93-5p mimic and inhibitor on odontogenic differentiation of hDPSCs. A pulpotomy rat model was further established, microCT and histological analyses were performed to explore the effects of KDM6B-overexpression and miR-93-5p inhibition on the formation of tertiary dentin.ResultsThe expression level of miR-93-5p decreased as odontoblast differentiated, in parallel with elevated expression of histone demethylase KDM6B. In hDPSCs, miR-93-5p overexpression inhibited the odontogenic differentiation and vice versa. MiR-93-5p targeted 3′ untranslated region (UTR) of KDM6B, thereby inhibiting its protein translation. Furthermore, KDM6B bound the promoter region of BMP2 to demethylate H3K27me3 marks and thus upregulated BMP2 transcription. In the rat pulpotomy model, KDM6B-overexpression or miR-93-5p inhibition suppressed H3K27me3 level in DPSCs and consequently promoted the formation of tertiary dentin.ConclusionsMiR-93-5p targets epigenetic regulator KDM6B and regulates H3K27me3 marks on BMP2 promoters, thus modulating the odontogenic differentiation of DPSCs and dentin formation.