[The Mechanism of GREM1's Effect on Osteogenic/Odontogenic Differentiation of Stem Cells from Apical Papilla].

OBJECTIVE: To study the effect of bone morphogenetic protein (BMP) antagonist Gremlin 1 (GREM1) on the function of stem cells from apical papilla (SCAPs) and explore its mechanism. METHODS: After isolation and culturing of stem cells from apical papilla in vitro, immunofluorescent staining was done to examine the subcellular localization of GREM1 in SCAPs. Transfection with lentiviral GREM1 shRNA was done to knock-down the GREM1. The SCAPs were subjected to osteogenic induction in both the GREM1 knockdown group and the control group, and the knockdown effect of GREM1 was examined using real time-PCR and Western blot. Two groups of cells were collected and the alkaline phosphatase (ALP) activity was measured 7 d after osteogenic induction. Alizarin red staining was done 3 weeks after osteogenic/odontogenic induction and real time-PCR was done after 0, 1, 2, 3 weeks of osteogenic induction to examine the expression of osteogenic/odontogenic marker genes, including osteocalcin ( OCN), osteopontin ( OPN), bone sialoprotein ( BSP), dentin matrix protein 1 ( DMP1), dentin sialophosphoprotein ( DSPP) and and the critical transcription factor osterix ( OSX), Runt-related transcription factor 2 ( RUNX2), and distal-less homebox 2 ( DLX2). Two groups of cells were collected, and CCK-8 and CFSE assay were used to evaluate changes in cell proliferation. In addition, real time-PCR was used to examine the expression of senescence-related genes p53 and wide-type activated factor 1 ( Waf1), a regulatory factor of the cell cycle, stemness associated gene krupple-like factor 4 ( KLF4), and SRY related HMG box-2 ( SOX2), and the expression of bone morphogenetic protein ( BMP) 2, 4, 5, 6, 7, 9 after GREM1 knockdown. RESULTS: Immunofluorescence staining showed that the expression of GREM1 in the nucleus was higher than that in the cytoplasm. Real time-PCR and Western blot affirmed that GREM1 was knocked down steadily. The ALP activity of the GREM1 knockdown group was higher than that of the control group ( P<0.05), and the alizarin red staining was lighter than that of the control group. The expression of OCN and DMP1 increased in the first, second and third week, OPN was increased in the second week, BSP increased in the third week, DSPP increased in the first week, and the difference was statistically significant ( P<0.05). The key osteogenic transcription factors RUNX2, OSX, and DLX2 all increased at different stages, and the difference was statistically significant ( P<0.05). CCK-8 and CFSE assay showed that the proliferation ability of the GREM1 knockdown group decreased ( P<0.05). In the GREM1 knockdown group, the expression of BMP2, 6, and 7 increased, the expression of SOX2 and KLF4 increased, while the expression of p53 and Waf1 decreased ( P<0.05). CONCLUSIONS: The knockdown of GREM1 enhanced the osteogenic/odontogenic differentiation and stemness of SCAPs and inhibited the proliferation and senescence of SCAPs. Effects of GREM1 on the function of SCAPs maybe achieved through regulating the gene expression of BMP2, BMP6, and BMP7 at the mRNA level.

Is PEGylated G-CSF superior to G-CSF in patients with breast cancer receiving chemotherapy? A systematic review and meta-analysis.

BACKGROUND: PEGylated granulocyte colony-stimulating factor (G-CSF) is a safe alternative to G-CSF to improve chemotherapy-induced neutropenia (CIN). This superiority has resulted in its increased use by physicians; however, the superiority of PEGylated G-CSF for CIN in breast cancer has not been conclusively determined. OBJECTIVES: To assess the superiority of PEGylated G-CSF for CIN in breast cancer in terms of effectiveness and safety via a systematic review and meta-analysis. METHODS: A literature search in PubMed, Embase, Cochrane Library, and Web of Science was performed for eligible studies published from database inception to December 2019. All studies comparing PEGylated G-CSF and G-CSF for CIN of breast cancer were reviewed. After literature selection, data extraction and quality assessment were performed by two reviewers independently. Meta-analysis was conducted using Revman, version 5.2. RESULTS: Nine randomized controlled trials were finally identified. The publication bias of these studies was acceptable. For the endpoint of effectiveness, analysis of the incidence/duration of grade ≥ 3 neutropenia, the duration of grade 4 neutropenia, the incidence of febrile neutropenia (FN), and the time to absolute neutrophil count recovery showed no advantage of PEGylated G-CSF over G-CSF for CIN of breast cancer (P > 0.05), with the premise of a sufficient dose of G-CSF according to the guidelines. No significant differences in grade 4 adverse events were observed between the groups (P = 0.29), and PEGylated G-CSF did not increase the incidence of skeletal and/or muscle pain compared with G-CSF (P = 0.32). CONCLUSION: PEGylated G-CSF was as effective and safe as G-CSF to reduce CIN in breast cancer but did not show an obvious superiority. However, in clinical practice, PEGylated G-CSF has an obvious advantage in terms of convenience, which could improve patient's quality of life.

Inflammatory myofibroblastic tumors of the nasal cavity and paranasal sinus: a clinicopathologic study of 25 cases and review of the literature.

Inflammatory myofibroblastic tumor (IMT) is rare in nasal cavity and paranasal sinus. The aim of this study was to describe the clinicopathological features of sinonasal IMT and analyze the relationship between the clinicopathological features and the prognosis. A retrospective study of 25 IMT patients between 2001 and 2012 was performed. Data on clinical features, treatment, and follow-up were recorded. The histological characters were observed. Overall survival (OS) and event-free survival (EFS) were estimated using the Kaplan-Meier method. Clinically, the most common symptoms were nasal obstruction, facial pain, and toothache. Twenty patients received follow-ups 6-120 months after initial diagnosis. Fifteen (75 %) developed recurrence 1 or more times. One patient had left cervical lymph node metastasis (5 %). Five patients died of the tumor (25 %). Histologically, the IMTs composed of bland spindle cells admixed with a prominent infiltrate of plasma cells and lymphocytes and showed obvious atypia in recurrent cases. Histology with necrosis, mitosis (≥1/10 HPF), ganglion-like cells, histological pattern I or II and relapse (≥4 times) was significantly associated with poor OS and EFS. IMT of the nasal cavity and paranasal sinuses exhibits relatively bland histologic appearances, but can shows strongly aggressive behavior and relatively poor outcomes. Multiple relapse, necrosis, frequent mitosis, the presence of ganglion-like cells, and histological pattern might be associated with poor clinical outcomes.

GREM1 Negatively Regulates Osteo-/Dentinogenic Differentiation of Dental Pulp Stem Cells via Association with YWHAH.

OBJECTIVE: To investigate the biological regulatory function of Gremlin1 (GREM1) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein eta (YWHAH) in dental pulp stem cells (DPSCs), and determine the underlying molecular mechanism involved. METHODS: Alkaline phosphatase (ALP) activity, alizarin red staining, scratch migration assays and in vitro and in vivo osteo-/dentinogenic marker detection of bone-like tissue generation in nude mice were used to assess osteo-/dentinogenic differentiation. Coimmunoprecipitation and polypeptide microarray assays were employed to detect the molecular mechanisms involved. RESULTS: The data revealed that knockdown of GREM1 promoted ALP activity, mineralisation in vitro and the expression of osteo-/dentinogenic differentiation markers and enhanced osteo-/ dentinogenesis of DPSCs in vivo. GREM1 bound to YWHAH in DPSCs, and the binding site was also identified. Knockdown of YWHAH suppressed the osteo-/dentinogenesis of DPSCs in vitro, and overexpression of YWHAH promoted the osteo-/dentinogenesis of DPSCs in vitro and in vivo. CONCLUSION: Taken together, the findings highlight the critical roles of GREM1-YWHAH in the osteo-/dentinogenesis of DPSCs.

Analysis of Risk Factors Associated with Early Childhood Caries.

Objective: To identify the main factors associated with early childhood caries by analyzing the risk factors of early childhood caries, thus providing a reference for developing prevention programs to reduce the risk of early childhood caries. Methods: We selected a total of 221 children aged 3-4 years from two kindergartens in Tongzhou District, Beijing for this study. We conducted oral examination and the caries activity test (Cariostat) on children and their parents / primary caregivers, and the parents / primary caregivers additionally answered a questionnaire survey. Based on the results, we comprehensively evaluated the caries status of children and statistically analyzed the caries-related factors to identify the relevant risk factors. Results: The mean age of children in the study children was 40.08 ± 2.65 months, with a caries prevalence rate of 54.97% and a mean caries value of 4.61. Early childhood caries was correlated with the intake frequency of sugary foods, intake of sugary foods before bedtime, frequency of tooth brushing, oral health knowledge of parents, caries susceptibility, and age of starting to brush teeth. Logistic regression analysis results showed that the intake frequency of sugary foods, oral health knowledge of parents, and caries susceptibility were the factors influencing early childhood caries, especially the intake frequency of sugary foods. Conclusion: The intake frequency of sugary foods, intake of sugary foods before bedtime, frequency of brushing teeth, oral health knowledge of parents, caries susceptibility, and age of starting to brush teeth were associated with early childhood caries. Among these, the intake frequency of sugary foods, oral health knowledge of parents, and caries susceptibility, especially the intake frequency of sugary foods, were the influencing factors.

Poly(acrylic acid)-modified Fe3O4 microspheres for magnetic-targeted and pH-triggered anticancer drug delivery.

Monodisperse poly(acrylic acid)-modified Fe(3)O(4) (PAA@Fe(3)O(4)) hybrid microspheres with dual responses (magnetic field and pH) were successfully fabricated. The PAA polymer was encapsulated into the inner cavity of Fe(3)O(4) hollow spheres by a vacuum-casting route and photo-initiated polymerization. TEM images show that the samples consist of monodisperse porous spheres with a diameter around 200 nm. The Fe(3)O(4) spheres, after modification with the PAA polymer, still possess enough space to hold guest molecules. We selected doxorubicin (DOX) as a model drug to investigate the drug loading and release behavior of as-prepared composites. The release of DOX molecules was strongly dependent on the pH value due to the unique property of PAA. The HeLa cell-uptake process of DOX-loaded PAA@Fe(3)O(4) was observed by confocal laser scanning microscopy (CLSM). After being incubated with HeLa cells under magnet magnetically guided conditions, the cytotoxtic effects of DOX-loaded PAA@Fe(3)O(4) increased. These results indicate that pH-responsive magnetic PAA@Fe(3)O(4) spheres have the potential to be used as anticancer drug carriers.

[Clinicopathologic study of sinonasal inflammatory myofibroblastic tumor].

OBJECTIVE: To study the clinicopathologic features, immunophenotype and ultrastructural features of sinonasal inflammatory myofibroblastic tumors (IMT). METHODS: The clinical and histologic features of 5 cases of sinonasal IMT were reviewed. Immunohistochemical study for vimentin, MSA, SMA, calponin, h-caldesmon, desmin, ALK, fibronectin, CK, S-100 and Ki-67 was carried out. Ultrastructural examination was also performed in two of the cases. RESULTS: The patients age ranged from 28 to 62 years (mean = 43 years). The male-to-female ratio was 2:3. The clinical presentation included nasal obstruction, nasal discharge, nasal bleeding, facial pain, facial swelling, toothache and tear overflow. All of the 5 patients suffered from disease relapses; and 4 of them had recurrences for more than 5 times. One patient had lymph node metastasis and 3 patients died of the disease. Histologically, the tumor cells were arranged in interlacing fascicles and sometimes haphazard in fashion. They were spindly in shape, cytoplasm eosinophilic with mild nuclear atypia and a low mitotic activity. The intervening stroma was myxoid in appearance accompanied by lymphocyte and plasma cell infiltration, abundant blood vessels and focal collagenized areas. In 3 of the recurrent cases, the tumor cells displayed increased nuclear atypia and mitotic activity (average about 5 to 6 per 10 high-power fields), accompanied by patchy necrosis, less inflammatory cell infiltration and focal sarcomatous changes. Immunohistochemical study showed that the tumor cells were diffusely positive for vimentin. SMA, MSA, calponin and fibronectin were variably expressed. Desmin was weakly positive in 1 case. The staining for h-caldesmon, ALK, S-100 and CK was negative. The Ki-67 proliferation index increased with tumor recurrences. Electron microscopy revealed abundant rough endoplasmic reticulum and dense body formation in the cytoplasm. There were an increased amount of collagen fibers in the stroma. CONCLUSIONS: IMT rarely occurs in nasal cavity and paranasal sinuses. The tumor is prone to local invasion and recurrences, with subsequent progression to frank malignancy and distant metastasis, resulting in high mortality and poor prognosis. Complete surgical resection remains the main modality of treatment.

Enriched trimethylation of lysine 4 of histone H3 of WDR63 enhanced osteogenic differentiation potentials of stem cells from apical papilla.

INTRODUCTION: Dental tissue-derived mesenchymal stem cells (MSCs) are a reliable cell source for dental tissue regeneration. However, the molecular mechanisms underlying their directed differentiation remain unclear, thus limiting their use. Trimethylation of lysine 4 of histone H3 (H3K4Me3) correlates with gene activation and osteogenic differentiation. We used stem cells from apical papilla (SCAPs) to investigate the effects of genomic changes in H3K4Me3 modification at gene promoter regions on MSC osteogenic differentiation. METHODS: ChIP-on-chip assays were applied to compare the H3K4Me3 profiles at gene promoter regions of undifferentiated and differentiated SCAPs. Alkaline phosphatase activity assay, alizarin red staining, quantitative analysis of calcium, the expressions of osteogenesis-related genes, and transplantation in nude mice were used to investigate the osteogenic differentiation potentials of SCAPs. RESULTS: In differentiated SCAPs, 119 gene promoters exhibited >2-fold increases of H3K4Me3; in contrast, the promoter regions of 21 genes exhibited >2-fold decreases of H3K4Me3. On the basis of enriched H3K4Me3 and up-regulated gene expression on the osteogenic differentiation of SCAPs, WDR63 may be a potential regulator for mediating SCAP osteogenic differentiation. Through gain-of-function and loss-of-function studies, we discovered that WDR63 enhances alkaline phosphatase activity, mineralization, and the expression of BSP, OSX, and RUNX2 in vitro. In addition, transplant experiments in nude mice confirmed that SCAP osteogenesis is triggered by activated WDR63. CONCLUSIONS: These results indicate that WDR63 is a positive enhancer for SCAP osteogenic differentiation and suggest that activation of WDR63 signaling might improve tissue regeneration mediated by MSCs of dental origin.

Bone morphogenetic protein 2-induced human dental pulp cell differentiation involves p38 mitogen-activated protein kinase-activated canonical WNT pathway.

Both bone morphogenetic protein 2 (BMP2) and the wingless-type MMTV integration site (WNT)/ß-catenin signalling pathway play important roles in odontoblast differentiation and dentinogenesis. Cross-talk between BMP2 and WNT/ß-catenin in osteoblast differentiation and bone formation has been identified. However, the roles and mechanisms of the canonical WNT pathway in the regulation of BMP2 in dental pulp injury and repair remain largely unknown. Here, we demonstrate that BMP2 promotes the differentiation of human dental pulp cells (HDPCs) by activating WNT/ß-catenin signalling, which is further mediated by p38 mitogen-activated protein kinase (MAPK) in vitro. BMP2 stimulation upregulated the expression of ß-catenin in HDPCs, which was abolished by SB203580 but not by Noggin or LDN193189. Furthermore, BMP2 enhanced cell differentiation, which was not fully inhibited by Noggin or LDN193189. Instead, SB203580 partially blocked BMP2-induced ß-catenin expression and cell differentiation. Taken together, these data suggest a possible mechanism by which the elevation of ß-catenin resulting from BMP2 stimulation is mediated by the p38 MAPK pathway, which sheds light on the molecular mechanisms of BMP2-mediated pulp reparative dentin formation.

Multifunctional core-shell structured nanocarriers for synchronous tumor diagnosis and treatment in vivo.

Multifunctional, mesoporous, silica-coated upconversion luminescent/magnetic NaGdF4:Yb/Er@NaGdF4:Yb@mSiO2-PEG (referred to as UCNPS; PEG=polyethylene glycol) nanocomposites were fabricated through a phase-transfer-assisted surfactant-templating coating process, followed by hydrophilic polymer (PEG) functionalization to improve the stability and biocompatibility. The UCNP core imparts the nanomaterials with luminescence and magnetic properties for simultaneous upconversion optical and magnetic resonance (MR) imaging, whereas the mesoporous shell affords the nanomaterials the ability to load the anticancer drug doxorubicin. Proof-of-principle in vitro and in vivo experiments are presented to demonstrate that the resultant composite nanomaterials can serve as nanotheranostics for synchronous upconversion luminescence/MR dual modal imaging and anticancer drug delivery; this finally realizes the integration of diagnostics and the treatment of cancers.

[Comparison of bonding properties of five adhesives in primary dentin].

OBJECTIVE: To evaluate the microtensile bond strength (µTBS) of five dentin adhesives and their respective fracture modes. METHODS: The flat dentine surfaces of 75 primary teeth were randomly divided into five groups,which was treated with FL-BondII(group A), Clearfil Protect Bond(group B), Clearfil SE Bond(group C), Adper(TM) Easy One(group D), and Single Bond 2(group E) respectively. The µTBS was determined with microtensile tester and the fracture mode was observed by scanning electron microscope(SEM). RESULTS: The mean µTBS for group A,B,C,D and E was (28.3 ± 2.2), (32.4 ± 2.5), (38.3 ± 2.8), (32.9 ± 3.4) and (23.2 ± 1.9) MPa respectively. There was significant difference between group C and group A,E (P < 0.01), and no significant difference between group C and group B,D. There was no significant difference between group A and group E (P > 0.05). The SEM indicated that there was no significant difference in the fracture mode. CONCLUSIONS: The bonding property of Clearfil Protect Bond is equivalent to Clearfil SE Bond and Adper(TM) Easy One, superior to Single Bond 2 and more suitable for primary dentin bonding .

[Effect of host derived matrix metalloproteinase on the degradation of root dentin collagen].

OBJECTIVE: To evaluate the effect of dentin matrix metalloproteinase (MMP) on the degradation of root dentin collagen. METHODS: Root dentin powder was demineralized with acetic acid (pH 4.0) at 4 degrees C for 14 d, then dialysed and centrifuged. Precipitation was divided into 7 groups, with 6 samples in each group, and each sample was 50.0 mg. One milliliter artificial saliva with a different reagent was added in each sample respectively. The reagents were 2 mmol/L APMA (MMP activator), 2 mmol/L EDTA, 100 mmol/L EDTA, 200 mmol/L EDTA, 0.2% and 0.02% chlorhexidine (MMP inhibitor), and the blank artificial saliva was taken as control. The amount of degraded collagen of each sample was determined with hydroxyproline assay kit. Scanning electron microscope was employed to observe the morphological and structural changes of root dentin which was demineralized or put into artificial saliva after being demineralized. RESULTS: The mean amount of degraded collagen in APMA group was significantly higher than that in the blank group (P < 0.05). The mean amount of degraded collagen in 2 mmol/L, 100 mmol/L, 200 mmol/L EDTA, 0.02% and 0.2% chlorhexidine groups was dramatically lower than that of the APMA group and the blank (P < 0.01). SEM observation indicated that the structural integrity of the collagen network on root surface dentin still existed in root dentin surface after being demineralized alone, while collagenous fibril was destructed and the structural integrity on root dentin surface disappeared after being demineralized and treated by artificial saliva. CONCLUSIONS: MMP in root dentin can degrade root dentin collagen after being activated at low pH followed by neutralization. The results suggest that host MMP may play an important role in the process of dentin caries formation.