The fusion of keratinized epithelium, an indication of early implant placement in the aesthetic area: an animal study.

BACKGROUND: In the period of the early implant placement, the socket is mainly occupied by provisional matrix (PM). Keratinized epithelium (KE) is critical for primary wound closure. Although both KE and PM are important, the detailed relationship among migrating KE, PM formation and indication of the early implant placement is still unclear. OBJECTIVE: This research aimed to locate a healing stage of KE with highest osteogenic PM formation after tooth extraction, which could be treated as the optimal time point for early implant placement. MATERIAL AND METHODS: Mice were sacrificed on days 1, 2, 3, 4 and 6 after incisor extraction. Clinical, histological, and immunohistochemical evaluations of the extraction sockets were performed, and statistical analyses were conducted. We then inserted implants into the PM with the greatest bioactivity and observed its osseointegration pattern for 3, 10, 17 and 30 days. RESULT: When KE fusion was reached, sockets were dominated by PM with the greatest expression of osteocalcin (OC, P < 0.05) and high levels of CD34 and Runx2. OC and Runx2 expression were positively correlated with KE coverage (P < 0.05). When the implant was inserted at 4 days' healing, the PM maintained its osteogenic ability, and osseointegration proceeded perfectly. CONCLUSION: The migration of KE was correlated with the formation of highly osteogenic and angiogenic PM. And the fusion of KE could be treated as an indication for early implant placement.

Osteoclast differentiation and dynamic mRNA expression during mice embryonic palatal bone development.

This study is the first to investigate the process of osteoclast (OCL) differentiation, its potential functions, and the associated mRNA and signalling pathways in embryonic palatal bone. Our findings suggest that OCLs are involved in bone remodelling, bone marrow cavity formation, and blood vessel formation in embryonic palatal bone. We observed TRAP-positive OCLs at embryonic day 16.5 (E16.5), E17.5, and E18.5 at the palatal process of the palate (PPP) and posterior and anterior parts of the palatal process of the maxilla (PPMXP and PPMXA, respectively), with OCL differentiation starting 2 days prior to TRAP positivity. By comparing the key periods of OCL differentiation between PPMX and PPP (E14.5, E15.5, and E16.5) using RNA-seq data of the palates, we found that the PI3K-AKT and MAPK signalling pathways were sequentially enriched, which may play critical roles in OCL survival and differentiation. Csf1r, Tnfrsff11a, Ctsk, Fos, Tyrobp, Fcgr3, and Spi1 were significantly upregulated, while Pik3r3, Tgfbr1, and Mapk3k7 were significantly downregulated, in both PPMX and PPP. Interestingly, Tnfrsff11b was upregulated in PPMX but downregulated in PPP, which may regulate the timing of OCL appearance. These results contribute to the limited knowledge regarding mRNA-specific steps in OCL differentiation in the embryonic palatal bone.

High throughput miRNA sequencing and bioinformatics analysis identify the mesenchymal cell proliferation and apoptosis related miRNAs during fetal mice palate development.

BACKGROUND: Palatogenesis requires a precise spatiotemporal regulation of gene expression. Recent studies indicate that microRNAs (miRNAs) are key factors in normal palatogenesis. The present study aimed to explain the regulatory mechanisms of miRNAs during palate development. METHODS: Pregnant ICR mice were choose at embryonic day 10.5 (E10.5). Hemotoxylin and eosin (H&E) staining was used to observe the morphological changes during the development of palatal process at embryonic day (E)13.5, E14.0, E14.5, E15.0 and E15.5. The fetal palatal tissues were collected at E13.5, E14.0, E14.5 and E15.0 to explore miRNA expression and function by high throughput sequencing and bioinformatic analysis. Mfuzz cluster analysis was used to look for miRNAs related to the fetal mice palate formation. The target genes of miRNAs were predicted by miRWalk. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed base on target genes. The mesenchymal cell proliferation and apoptosis related miRNAs-genes networks were predicted and constructed using miRWalk and Cytoscape software. The expression of mesenchymal cell proliferation and apoptosis related miRNAs at the E13.5, E14.0, E14.5, and E15.0 was detected by a quantitative real-time PCR (RT-qPCR) assay. RESULTS: H&E staining found that the palatal process grows vertically along the sides of the tongue at E13.5, the position of the tongue begins to descend and the bilateral palatal processes rise above the tongue at E14.0, the palatal process grows horizontally at E14.5, there is palatal contact fusion at E15.0, and the palatal suture disappeared at E15.5. Nine clusters of miRNA expression changes were identified in the fetal mice palate formation progression, including two reducing trends, two rising trends and five disordered trends. Next, the heatmap showed the miRNA expression from Clusters 4, 6, 9, 12 in the E13.5, E14.0, E14.5 and E15.0 groups. GO functional and KEGG pathway enrichment analysis found target genes of miRNAs in clusters involved in regulation of mesenchymal phenotype and the mitogen-activated protein kinase (MAPK) signaling pathway. Next, mesenchymal phenotype related miRNA-genes networks were constructed. The heatmap showing that the mesenchymal phenotype related miRNA expression of Clusters 4, 6, 9 and 12 at E13.5, E14.0, E14.5 and E15.0. Furthermore, the mesenchymal cell proliferation and apoptosis related miRNA-gene networks were identified in Clusters 6 and 12, including mmu-miR-504-3p-Hnf1b, etc. The expression level of mesenchymal cell proliferation and apoptosis related miRNAs at the E13.5, E14.0, E14.5, and E15.0 was verified by a RT-qPCR assay. CONCLUSIONS: For the first time, we identified that clear dynamic miRNA expression during palate development. Furthermore, we demonstrated that mesenchymal cell proliferation and apoptosis related miRNAs, genes and the MAPK signaling pathway are important during fetal mice palate development.

Effect of Eleutheroside E on an MPTP-Induced Parkinson's Disease Cell Model and Its Mechanism.

This research investigated the effects of eleutheroside E (EE) on the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease cell model and its mechanism. Methods: To create a cell model of Parkinson's disease, MPTP (2500 µmol/L) was administered to rat adrenal pheochromocytoma cells (PC-12) to produce an MPTP group. Selegiline (50 µmol/L) and MPTP had been administered to the positive group beforehand. The eleutheroside E group was divided into low-, medium-, and high-concentration groups, in which the cells were pretreated with eleutheroside E at concentrations of 100 µmol/L, 300 µmol/L, and 500 µmol/L. Next, MPTP was added to the cells separately. The CCK-8 method was used to measure the cell survival rate. Apart from the CCK-8 method, mitochondrial membrane potential detection, cell reactive oxygen species (ROS) detection, and other methods were also adopted to verify the effect of low, medium, and high concentrations of eleutheroside E on the MPTP-induced cell model. Western blot analysis was used to detect changes in the expression of intracellular proteins CytC, Nrf2, and NQO1 to clarify the mechanism. The results are as follows. Compared with the MPTP group, the survival rates of cells at low, medium, and high concentrations of eleutheroside E all increased. The mitochondrial membrane potential at medium and high concentrations of eleutheroside E increased. The ROS levels at medium and high concentrations of eleutheroside E decreased. Moreover, the apoptosis rate decreased and the expression levels of the intracellular proteins CytC, Nrf2, and NQO1 were upregulated. Conclusion: Eleutheroside E can improve the MPTP-induced apoptosis of PC-12 cells by increasing the mitochondrial membrane potential and reducing the level of intracellular reactive oxygen species (ROS). Moreover, the apoptosis of cells is regulated by the expression of CytC, Nrf2, and NQO1 proteins.

Development of a food preservative from sea buckthorn together with chitosan: Application in and characterization of fresh-cut lettuce storage.

The purpose was to create a novel composite food preservative for fresh-cut lettuce using flavonoids and chitosan from sea buckthorn leaves (SBL). Sea buckthorn leaves were extracted with ethanol as the extraction solvent and ultrasonic-assisted extraction to obtain flavonoid from sea buckthorn leaf crude (FSL), and then the FSL was secondarily purified with AB-8 resin and polyamide resin to obtain flavonoid from sea buckthorn leaf purified (FSL-1). Different concentrations of FSL-1 and chitosan were made into a composite preservative (FCCP) by magnetic stirring and other methods, containing 1% chitosan preservative (CP) alone, 0.5-2 mg/ml of FSL-1 and 1% chitosan composite preservative (FCCP-1, FCCP-2, FCCP-3, and FCCP-4), and the FSL-1 concentrations were analyzed the effect of FSL-1 concentration on the physicochemical properties of the composite preservatives, including their film-forming ability, antioxidant capacity and ability to prevent bacterial growth, was analyzed. To further investigate the effect of the combined preservatives on fresh-cut lettuce, different FCCPs were applied to the surface was stored at 4°C for 7 days. Then the changes in weight loss, hardness, browning index, total chlorophyll content, SOD and MDA were analyzed. It was used to assess the physicochemical indicators of fresh-cut lettuce throughout storage. According to the results of Fourier transform infrared spectroscopy, FSL-1 and chitosan interacted to form hydrogen bonds, and the contact angle and viscosity of FCCP increased on both horizontal glass and polystyrene plates, indicating the good film-forming properties of the composite preservation solution. With the diameter of the antibacterial zone of Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, and Listeria monocytogenes being (21.39 ± 0.22), (17.43 ± 0.24), (15.30 ± 0.12), and (14.43 ± 0.24) mm, respectively. It was proved that the antibacterial activity of FCCP became stronger with the increase of FSL-1 concentration and had the best antibacterial effect on S. aureus. The complex preservative showed the best scavenging effect on ferric reducing antioxidant capacity, DPPH radicals (96.64%) and 2,2'-Azinobis- (3-ethylbenzthiazoline-6-sulphonate) (ABTS) radicals (99.42%) when FSL-1 was added at 2 mg/ml. When fresh-cut lettuce was coated with FCCP for the same storage time, various indicators of lettuce such as weight loss, hardness, browning index, SOD activity and MDA content were better than the control group showing good potential in fresh-cut vegetables and fruits preservation. FCCP holds great promise for food safety quality and shelf-life extension as a new natural food preservative. The waste utilization of sea buckthorn leaves can greatly improve his utilization and economic benefits.

Preparation and characterization of geraniol nanoemulsions and its antibacterial activity.

Geraniol nanoemulsions (G-NE) based on Tween 80 and medium chain triglyceride (MCT) as surfactant and co-surfactant, respectively, has been prepared by the spontaneous emulsification method. Its physical and chemical properties such as mean particle size, zeta potential, PDI, pH, viscosity, contact angle, appearance morphology, and stability (storage stability, thermal stability, centrifugal properties, acid-base stability, and freeze-thaw properties) of the droplet were analyzed. The results showed that the mean particle size of G-NE was 90.33 ± 5.23 nm, the PDI was 0.058 ± 0.0007, the zeta potential was -17.95 ± 5.85 mV and the encapsulation efficiency was >90%. The produced G-NE has been demonstrated to be fairly stable in long-term storage at 4°C, pH = 5 and high-speed centrifuges. Moreover, G-NE had a significant inhibition effect on Staphylococcus aureus, Escherichia coli, Salmonella typhimurium and Listeria monocytogenes (p < 0.05). The bacterial inhibition rates of G-NE at a concentration of 1 MIC were 48, 99, 71.73, and 99% after 12 h of action against these four foodborne pathogenic bacteria, respectively. Therefore, the results obtained indicated that nanoemulsification enhanced the stability and antibacterial activity of geraniol to some extent, which will promote the utilization of geraniol in food preservation.

Protective Effect of Flavonoids from Mulberry Leaf on AAPH-Induced Oxidative Damage in Sheep Erythrocytes.

To evaluate the antioxidant activity of flavonoids extracted from Chinese herb mulberry leaves (ML), flavonoids from mulberry leaves (FML) were extracted and purified by using ultrasonic-assisted enzymatic extraction and D101 macroporous resin. Using LC-MS/MS-Liquid Chromatography with tandem mass spectrometry analysis, hesperidin, rutoside, hyperoside, cyanidin-3-o-glucoside, myricitrin, cyanidin, and quercetin were identified, and NMR and UV were consistent with the verification of IR flavonoid characteristics. The antioxidant activity of FML has also been evaluated as well as the protective effect on 2,2 0-azobis (2-amidinopropane) dihydrochloride (AAPH)-induced oxidative stress. The results showed that FML exhibited powerful antioxidant activity. Moreover, FML showed dose-dependent protection against AAPH-induced sheep erythrocytes' oxidative hemolysis. In the enzymatic antioxidant system, pretreatment with high FML maintained the balance of SOD, CAT, and GSH-Px; in the non-enzymatic antioxidant system, the content of MDA can be effectively reduced after FML treatment. This study provides a research basis for the development of natural products from mulberry leaves.

Neuroprotection of Gastrodia elata polyphenols against H2O2-induced PC12 cell cytotoxicity by reducing oxidative stress.

It has been suggested that oxidative stress (OS) has a role in the development of aging and neurodegenerative disorders. Biological molecules are easily damaged by reactive oxygen species, which can ultimately result in necrotic or apoptotic cell death. Foods containing phytochemicals, such as phenolic compounds, may have potential preventive effects against several diseases, including alzheimer's disease (AD), according to epidemiological and in vitro research. Gastrodia elata is a well-known homology of medicine and food plant that has been used for centuries in China and other East Asian countries to treat central nervous system disorders. In this study, we focused on the potential of the extract, Gastrodia elata polyphenols (GPP), for the prevention and treatment of AD. H2O2 induced PC12 cell damage was used to simulate the oxidative stress of AD. The effects of GPP on the injury model were evaluated by cell survival rate, lactate dehydrogenase (LDH), lipid peroxidation (MDA), production of intracellular antioxidant enzymes, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), cellular inflammation level and apoptosis level. The results showed that GPP pretreatment had a protective effect by increasing cell viability, reducing lactate dehydrogenase infiltration, decreasing MDA and increasing intracellular antioxidant enzymes, diminishing reactive oxygen species production and decreasing mitochondrial membrane potential, reducing cell inflammation and decreasing apoptosis. Accordingly, it is suggested that GPP possessed promising neuroprotective benefits which enabled the prevention or therapeutic implementation of AD along with serving as a reference towards the exploitation of functional foods or drugs derived from Gastrodia elata.

Cellular Antioxidant Properties of Ischnoderma Resinosum Polysaccharide.

A predominant polysaccharide isolated from Ischnoderma resinosum underwent evaluation for its capacity to scavenge free radicals and its potential antioxidant properties at a cellular-oriented level. This proved that Ischnoderma resinosum polysaccharide (IRP) remarkably curtailed AAPH-induced erythrocyte hemolysis through the inhibition of the generation of ROS (p < 0.05). Rather, it caused the restoration of intracellular antioxidant enzyme (SOD, GSH-Px, and CAT) activities at an acceptable pace and the silencing of intracellular MDA formation, as well as the rescaling of LDH leakage. Furthermore, a model of oxidative stress in HepG2 cells was established by adopting 400 µM of hydrogen peroxide, which suggested that IRP manifests promising antioxidant activity. Notably, after the intervention of IRP in the H2O2-induced HepG2 cells, there was a statistical elevation in cell survivability (p < 0.05). IRP diminished the morphological alterations in the nucleus and decreased the secretion of ROS (p < 0.05), with a dose-dependent abrogation of apoptosis (p < 0.05). Consequently, IRP, which was isolated and purified, was able to scavenge free radicals and possessed favorable antioxidant activity that could dampen the occurrence of oxidative stimulation and effectively alleviate the AAPH-induced erythrocyte hemolysis and H2O2-induced oxidative damage in HepG2 cells. This provides a basis and theoretical reference for the development and utilization of IRP as a natural antioxidant, with emphasis on the exploitation of environmentally friendly and cost-effective antioxidants.

Revitalizing mouse diphyodontic dentition formation by inhibiting the sonic hedgehog signaling pathway.

BACKGROUND: Tooth regeneration depends on the longevity of the dental epithelial lamina. However, the exact mechanism of dental lamina regression has not yet been clarified. To explore the role of the Sonic hedgehog (Shh) signaling pathway in regression process of the rudimentary successional dental lamina (RSDL) in mice, we orally administered a single dose of a Shh signaling pathway inhibitor to pregnant mice between embryonic day 13.0 (E13.0) and E17.0. RESULTS: We observed that the Shh signaling pathway inhibitor effectively inhibited the expression of Shh signaling pathway components and revitalized RSDL during E15.0-E17.0 by promoting cell proliferation. In addition, mRNA-seq, reverse transcription plus polymerase chain reaction (RT-qPCR), and immunohistochemical analyses indicated that diphyodontic dentition formation might be related to FGF signal up-regulation and the Sostdc1-Wnt negative feedback loop. CONCLUSIONS: Overall, our results indicated that the Shh signaling pathway may play an initial role in preventing further development of mouse RSDL in a time-dependent manner.

Osteogenic and angiogenic profiles of the palatal process of the maxilla and the palatal process of the palatine bone.

Hard palate consists anteriorly of the palatal process of the maxilla (ppmx) and posteriorly of the palatal process of the palatine (ppp). Currently, palatal osteogenesis is receiving increasing attention. This is the first study to provide an overview of the osteogenesis process of the mouse hard palate. We found that the period in which avascular mesenchymal condensation becomes a vascularized bone structure corresponds to embryonic day (E) 14.5 to E16.5 in the hard palate. The ppmx and ppp differ remarkably in morphology and molecular respects during osteogenesis. Osteoclasts in the ppmx and ppp are heterogeneous. There was a multinucleated giant osteoclast on the bone surface at the lateral-nasal side of the ppmx, while osteoclasts in the ppp were more abundant and adjacent to blood vessels but were smaller and had fewer nuclei. In addition, bone remodeling in the hard palate was asymmetric and exclusively occurred on the nasal side of the hard palate at E18.5. During angiogenesis, CD31-positive endothelial cells were initially localized in the surrounding of palatal mesenchymal condensation and then invaded the condensation in a sprouting fashion. At the transcriptome level, we found 78 differentially expressed genes related to osteogenesis and angiogenesis between the ppmx and ppp. Fifty-five related genes were up/downregulated from E14.5 to E16.5. Here, we described the morphogenesis and the heterogeneity in the osteogenic and angiogenic genes profiles of the ppmx and ppp, which are significant for subsequent studies of normal and abnormal subjects.

Defining the critical period of hedgehog pathway inhibitor-induced cranial base dysplasia in mice.

BACKGROUND: The hedgehog signaling pathway is critical for developmental patterning of the limb, craniofacial and axial skeleton. Disruption of this pathway in mice leads to a series of structural malformations, but the exact role and critical period of the Hh pathway in the early development of the cranial base have been rarely described. RESULTS: Embryos exposed to vismodegib from E7.5, E9.5, and E10.5 had a higher percentage of cranial base fenestra. The peak incidence of hypoplasia in sphenoid winglets and severe craniosynostosis in cranial base synchondroses was observed when vismodegib was administered between E9.5 and E10.5. Cranial base craniosynostosis results from accelerating terminal differentiation of chondrocytes and premature osteogenesis. CONCLUSIONS: We define the critical periods for the induction of cranial base deformity by vismodegib administration at a meticulous temporal resolution. Our findings suggest that the Hh pathway may play a vital role in the early development of the cranial base. This research also establishes a novel and easy-to-establish mouse model of synostosis in the cranial base using a commercially available pathway-selective inhibitor.

Defining a critical period in calvarial development for Hedgehog pathway antagonist-induced frontal bone dysplasia in mice.

The Hedgehog (Hh) signalling pathway is essential for cellular proliferation and differentiation during embryonic development. Gain and loss of function of Hh signalling are known to result in an array of craniofacial malformations. To determine the critical period for Hh pathway antagonist-induced frontal bone hypoplasia, we examined patterns of dysmorphology caused by Hh signalling inhibition. Pregnant mice received a single oral administration of Hh signalling inhibitor GDC-0449 at 100 mg•kg-1 or 150 mg•kg-1 body weight at preselected time points between embryonic days (E)8.5 and 12.5. The optimal teratogenic concentration of GDC-0449 was determined to be 150 mg•kg-1. Exposure between E9.5 and E10.5 induced frontal bone dysplasia, micrognathia and limb defects, with administration at E10.5 producing the most pronounced effects. This model showed decreased ossification of the frontal bone with downregulation of Hh signalling. The osteoid thickness of the frontal bone was significantly reduced. The amount of neural crest-derived frontal bone primordium was reduced after GDC-0449 exposure owing to a decreased rate of cell proliferation and increased cell death.