In vivo study on osteogenic efficiency of nHA/ gel porous scaffold with nacre water-soluble matrix.

BACKGROUND/PURPOSE: Nano-hydroxyapatite (nHA)/ gel porous scaffolds loaded with WSM carriers are promising bone replacement materials that can improve osseointegration ability. This investigation aimed to evaluate the osteoinductive activity by implanting the composition of nano-hydroxyapatite (nHA)/ Gel porous scaffolds as a carrier of WSM via an animal model. MATERIALS AND METHODS: WSM was extracted and nHA was added to the matrix to construct porous composite scaffolds. The dose-effect curve of WSM concentration and alkaline phosphatase (ALP) activity was made by culturing rat osteoblasts and examining the absorbance. Three different materials were implanted into critical size defects (CSD) in the skulls of rats, which were further divided into four groups: WSM nHA /Gel group, n-WSM nHA /Gel group, HA powder group, and control group. RESULTS: WSM (150 µg/mL-250µg/mL) effectively improved the activity of ALP in rat osteoblasts. All rats in each group had normal healing. WSM-loaded nHA /Gel group showed better performance on newly-formed bone tissue of rat skull and back at 4th week and 8th week, respectively. At the 4th week, the network of woven bone formed in the WSM-loaded nHA/Gel scaffold material. At 8th week, the reticular trabecular bone in the WSM-loaded scaffold material became dense lamellar bone, and the defect was mature lamellar bone. In the subcutaneous implantation experiment, WSM-loaded nHA/Gel scaffold material showed a better performance of heterotopic ossification than the pure nHA/Gel scaffold material. CONCLUSION: WSM promotes osteoblast differentiation and bone mineralization. The results confirm that the nHA/ Gel Porous Scaffold with Nacre Water-Soluble Matrix has a significant bone promoting effect and can be used as a choice for tissue engineering to repair bone defects.

Network pharmacology-based strategy combined with molecular docking to explore the potential mechanism of agarwood against recurrent aphthous stomatitis.

To explore the antiinflammatory mechanism of agarwood on recurrent aphthous stomatitis (RAS). RAS is the most common mucosal disease in the oral cavity. The clinical application of traditional Chinese medicine found that agarwood has significant curative effect on peptic ulcer, but the effect and mechanism of agarwood on RAS remain unclear. This study is intended to predict the potential antiinflammatory mechanisms by which agarwood acts on RAS through network pharmacology and molecular docking. TCMSP database was used to screen the active components of agarwood. RAS targets were screened in Genecards, DisGeNET, and OMIM database. Venny, an online tool, screens for interacting genes between the two. Cytoscape software was used to construct the gene regulation map of active compounds target of agarwood. String Database building protein-protein interaction network. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathways were enriched in DAVID database. The key active ingredients and core targets were further verified by molecular docking. There were 9 effective compounds and 186 target genes in agarwood; RAS has 793 target genes. There were 41 interacting genes between agarwood and RAS. Interleukin 6, tumor necrosis factor, interleukin 1 beta, and cellular component motif ligand 2 may be key targets. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses predicted multiple pathways associated with RAS. Molecular docking results showed that the active compounds of agarwood combined well and stably with the target. The Chinese herbal medicine agarwood can relieve the inflammation of RAS through multiple targets and various ways. Its active compounds may be nominated as candidates for antiinflammatory drugs of RAS.

Regulation of the AGEs-induced inflammatory response in human periodontal ligament cells via the AMPK/NF-κB/ NLRP3 signaling pathway.

The heightened prevalence and accelerated progression of periodontitis in individuals with diabetes is primarily attributed to inflammatory responses in human periodontal ligament cells (HPDLCs). This study is aimed at delineating the regulatory mechanism of nucleotide-binding oligomerization domain-like receptors (NLRs) in mediating inflammation incited by muramyl dipeptide (MDP) in HPDLCs, under the influence of advanced glycation end products (AGEs), metabolic by-products associated with diabetes. We performed RNA-seq in HPDLCs induced by AGEs treatment and delineated activation markers for the receptor of AGEs (RAGE). It showed that advanced glycation end products modulate inflammatory responses in HPDLCs by activating NLRP1 and NLRP3 inflammasomes, which are further regulated through the NF-κB signaling pathway. Furthermore, AGEs synergize with NOD2, NLRP1, and NLRP3 inflammasomes to augment MDP-induced inflammation significantly.

Network pharmacology and molecular docking-based investigation on traditional Chinese medicine Astragalus membranaceus in oral ulcer treatment.

To analyze the mechanism of Astragalus membranaceus (AM) in molecular level in the oral ulcer (OU) treatment with reference to network pharmacology. Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform database was used in screening the AM active components and AM action targets; GeneCards database was used to screen OU targets; the common target were screened by Venny online tool; Cytoscape software was applied to construct the target gene regulation map of AM active components; STRING database was used to construct the protein-protein interaction network and the key targets were screened as per degree value; gene ontology enrichment and KEGG pathway enrichment of interactive genes were calculated through David database. There were 17 active ingredients and 429 target spots in Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform database. There are 606 target genes for OU in GeneCards database. There are 67 common targets, including 10 key targets: IL10, IL6, TNF, IL1B, CXCL8, CCL2, TLR4, IL4, ICAM1, and IFNG. It involves 30 gene ontology terms and 20 KEGG signal channels. The molecular docking results showed that quercetin and kaempferol had a good binding activity with IL6, IL1B, TNF, and CCL2. Network pharmacological analysis shows that AM can regulate multiple signal pathways through multiple targets to treat OU.

Salivary Pro-Inflammatory Markers and Smoking Status Influences the Treatment Effectiveness of Periodontal Disease Patients with Hypertension.

BACKGROUND: Hypertension and periodontal diseases share several risk factors. Inflammation biomarkers in saliva are related to hypertension and periodontal disease. The aim of this study was to explore the role of the salivary inflammatory biomarkers in the treatment effectiveness of patients with hypertension and periodontal disease. METHODS: This observational study enrolled 160 subjects diagnosed with periodontitis, 40 of which had a history of hypertension. All subjects had completed scaling and root planning therapeutic procedures within four weeks. The clinical periodontal parameters (i.e., bleeding on probing, plaque control record (PCR), and probing depth (PD)) were evaluated before and after the treatment. Pro-inflammatory markers were determined using a commercial kit. RESULTS: The recovery rate (PD 4-9 mm) in non-hypertensive subjects was significantly higher than in hypertensive subjects (60.47% vs. 52.60%, respectively; p = 0.04). All clinical parameters, excluding PCR, positively correlated with salivary IL-1ß at baseline and after completing treatment. Our results showed that increased salivary IL-1ß levels were positively associated with decreased PCR (ß = -27.65 and p = 0.05) and PD recovery rate (ß = -17.05 and p = 0.02) in hypertensive subjects. CONCLUSIONS: The present study sheds important light on the clinical use of salivary pro-inflammatory cytokines as valuable biomarkers for predicting the treatment effectiveness of patients suffering from hypertension and periodontitis.

Mining TCGA database for prognostic genes in head and neck squamous cell carcinoma microenvironment.

BACKGROUND/PURPOSE: Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignant tumors. The aim of this study was to elucidate the effect of tumor microenvironment-related genes on the prognosis of HNSCC and to obtain tumor microenvironment-related genes that can predict poor prognosis in HNSCC patients. MATERIALS AND METHODS: The ESTIMATE algorithm was applied to the HNSCC transcriptomic data downloaded from the TCGA (The cancer genome atlas), and then the samples were divided into two groups: high and low immune scoring groups, and high and low basal scoring groups to screen for differentially expressed genes (DEGs) associated with poor patient outcomes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to explore the potential functions of DEGs, and then to explore the potential prognostic value of individual DEGs. The results of survival analysis between DEGs and overall survival (OS) to explore tumor microenvironment-related genes relevant to the prognosis of HNSCC patients. RESULTS: Fifty-nine tumor microenvironment-related genes were screened for association of OS with HNSCC (P < 0.05). The GO and KEGG enrichment analysis showed that the selected DEGs may mediate immune response, extracellular matrix, and immunoglobulin binding via neutrophil activation in HNSCC. Six of these DEGs, GIMAP6, SELL, TIFAB, KCNA3, P2RY8 and CCR4 were most significantly associated with OS (P < 0.001). CONCLUSION: We identified six tumor microenvironment-related genes that were significantly associated with poor prognosis in HNSCC. These genes may inspire researchers to discover new targets and approaches for HNSCC treatment.

Advanced glycosylated end products restrain the osteogenic differentiation of the periodontal ligament stem cell.

BACKGROUND/PURPOSE: Many studies have confirmed that periodontal disease interacts with diabetes. The aim of this study was to examine whether the advanced glycosylated end products (AGEs), which are generated by diabetics, have important effects on the osteogenic differentiation of periodontal ligament stem cells (PDLSCs). MATERIALS AND METHODS: In this study PDLSCs were isolated from the periodontal ligaments of extracted third molar teeth. The subjects were divided into two groups, which included the normal control group (N-PDLSCs) and the AGEs-stimulating group (A-PDLSCs). Changes of receptor of AGEs (RAGE) and cumulative ROS in PDLSCs were monitored by western blot and flow cytometry, respectively. RESULTS: In the study AGEs noticeably inhibited the osteogenic differentiation of PDLSCs, with significant lower calcification nodules detected in A-PDLSCs (P < 0.01). RAGE expression level and ROS accumulation in A-PDLSCs were clearly higher than those in N-PDLSCs (P < 0.01). CONCLUSION: Our conclusions were that AGEs may cause the apoptosis of stem cells, which could lead to the disorder of bone differentiation function of PDLSCs.

The novel thiosemicarbazone, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), inhibits neuroblastoma growth in vitro and in vivo via multiple mechanisms.

BACKGROUND: Neuroblastoma is a relatively common and highly belligerent childhood tumor with poor prognosis by current therapeutic approaches. A novel anti-cancer agent of the di-2-pyridylketone thiosemicarbazone series, namely di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), demonstrates promising anti-tumor activity. Recently, a second-generation analogue, namely di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), has entered multi-center clinical trials for the treatment of advanced and resistant tumors. The current aim was to examine if these novel agents were effective against aggressive neuroblastoma in vitro and in vivo and to assess their mechanism of action. METHODS: Neuroblastoma cancer cells as well as immortalized normal cells were used to assess the efficacy and selectivity of DpC in vitro. An orthotopic SK-N-LP/Luciferase xenograft model was used in nude mice to assess the efficacy of DpC in vivo. Apoptosis in tumors was confirmed by Annexin V/PI flow cytometry and H&E staining. RESULTS: DpC demonstrated more potent cytotoxicity than Dp44mT against neuroblastoma cells in a dose- and time-dependent manner. DpC significantly increased levels of phosphorylated JNK, neuroglobin, cytoglobin, and cleaved caspase 3 and 9, while decreasing IkBα levels in vitro. The contribution of JNK, NF-ĸB, and caspase signaling/activity to the anti-tumor activity of DpC was verified by selective inhibitors of these pathways. After 3 weeks of treatment, tumor growth in mice was significantly (p < 0.05) reduced by DpC (4 mg/kg/day) given intravenously and the agent was well tolerated. Xenograft tissues showed significantly higher expression of neuroglobin, cytoglobin, caspase 3, and tumor necrosis factor-α (TNFα) levels and a slight decrease in interleukin-10 (IL-10). CONCLUSIONS: DpC was found to be highly potent against neuroblastoma, demonstrating its potential as a novel therapeutic for this disease. The ability of DpC to increase TNFα in tumors could also promote the endogenous immune response to mediate enhanced cancer cell apoptosis.

Genetically modified "obligate" anaerobic Salmonella typhimurium as a therapeutic strategy for neuroblastoma.

BACKGROUND: Neuroblastoma currently has poor prognosis, therefore we proposed a new strategy by targeting neuroblastoma with genetically engineered anaerobic Salmonella (Sal-YB1). METHODS: Nude and nonobese diabetic-severe combined immunodeficiency (NOD-SCID) orthotopic mouse models were used, and Sal-YB1 was administered via tail vein. The therapeutic effectiveness, bio-safety, and mechanisms were studied. RESULTS: No mice died of therapy-related complications. Tumor size reduction was 70 and 30% in nude and NOD-SCID mice, respectively. No Salmonella was detected in the urine; 75% mice had positive stool culture if diaminopimelic acid was added, but all turned negative subsequently. Tumor tissues had more Sal-YB1 infiltration, necrosis, and shrinkage in Sal-YB1-treated mice. Significantly higher expression of TLR4, TNF-stimulated gene 6 protein (TSG6), and cleaved caspase 1, 3, 8, and 9 was found in the tumor masses of the Sal-YB1-treated group with a decrease of interleukin 1 receptor-associated kinase (IRAK) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα). There was a high release of TNFα both in human macrophages and mouse tumor tissues with Sal-YB1 treatment. The antitumor effect of the supernatant derived from macrophages treated with Sal-YB1 could be reversed with TNFα and pan-caspase inhibitors. CONCLUSIONS: This new approach in targeting neuroblastoma by bio-engineered Salmonella with the assistance of macrophages indirectly may have a clinical therapeutic impact in the future.

[Clinicopathological characteristics of malignant transformation in 85 cases of oral leukoplakia].

OBJECTIVE: To investigate the histological features of oral leukoplakia which underwent malignant transformation and its correlation with the clinical manifestation. METHODS: A total of 1832 cases of oral leukoplakia were reviewed and the clinicopathological characteristics of malignant transformation were analyzed. RESULTS: Malignant transformation occurred in 85 cases (4.6%) of the 1832 cases. Thirty cases (2.1%) of 1404 cases with simple epithelial hyperplasia had malignant transformation. Fifty-five cases (12.9%) in 428 cases with epithelial dysplasia were transformed to malignancy, especially in the cases with moderate or severe dysplasia, in which the ratio of malignant transformation was higher than in the cases with simple epithelial hyperplasia (P < 0.005). Clinical parameters associated with an increased risk of malignant transformation were female gender and epithelial dysplasia was more often seen in non-homogenous leukoplakias than in homogenous (P < 0.005). CONCLUSIONS: Non-dysplastic leukoplakia may become malignant. Epithelial dysplasia was associated with an increased risk of malignant transformation. Leukoplakia in female may be at a higher risk for malignant transformation.

[Expression of integrin-linked kinase in oral squamous cell cancinoma and its significance].

OBJECTIVE: To investigate the expression patterns of integrin-linked kinase (ILK) in oral squamous cell carcinoma (OSCC). METHODS: The expression of ILK in 170 OSCC cases was examined by immunohistochemistry and periodic acid-schiff histochemistry. RESULTS: ILK immunoreactivity occurred in cancer cells and(or) stroma encircling the malignant tissue. Along with the deteriorated histopathologic grade of oral cancer, there was stepwise aberration exhibiting from ILK-negative, to ILK-positive in epithelia, to ILK-positive in both epithelia and stroma, and to ILK-positive in stroma. Through non-parameter test, in both lymphatic invasion [ILK-positive in stroma in well-differentiated, moderately-differentiated and poorly-differentiated is 2% (1/50), 18% (6/33) and 64% (18/28), respectively] and non-lymphatic invasion group [ILK-positive in stroma in well-differentiated, moderately-differentiated and poorly-differentiated is 16% (4/25), 9/18 and 10/16, respectively), the expression patterns of ILK exhibited significant correlations with histopathology (F = 17.742, P < 0.001, F = 4.394, P = 0.017). ILK expression between lymphatic metastasis and non-lymphatic metastasis group was significantly different (χ(2) = 14.418, P = 0.002). CONCLUSIONS: ILK inclines to express in stroma during the OSCC progress.

[Expression of integrin-linked kinase in oral leukoplakia and early invasive carcinoma].

OBJECTIVE: To investigate the transmutation and significance of integrin-linked kinase (ILK) in oral leukoplakia and early invasive squamous cell carcinoma. METHODS: Immunohistochemistry accompanied with periodic acid Schiff reaction (PAS) histochemistry was applied to detect the expression of ILK in 19 normal oral mucosa, 43 simple hyperplasia, 84 epithelial dysplasia, 54 early invasive carcinoma. RESULTS: Among the oral leukoplakia and early invasive squamous cell carcinoma, dramatic positive expression of ILK was found in 163/181 (90%), but was negative in normal mucosa. ILK staining in stroma exhibited a significantly direct correlation with the exacerbated epithelial dysplasia (chi(2) = 41.585, P < 0.001). Along with the oral carcinogenesis, there was an obvious tendency of change-overed ILK staining from superfacial to the basal orientation, the stratum basale diverted into positive, and compared with scattering in cytoplasm, the location of ILK was prone to cytomembrane in epithelium. In severe dysplasia and carcinoma in situ, there was a positive correlation between the basal layer and stroma expression of ILK (P = 0.029). It was not only a weaker tincture of ILK in cancer nest than lesional epithelia, but also an augmentation of stroma staining in early invasive squamous cell carcinoma [76% (41/54)] by contrast with severe dysplasia [45% (18/40)]. CONCLUSIONS: The pivotal role of ILK in the regulation of oral carcinogenesis is considerable, however, the precise mechanisms through which ILK affects cellular processes remain to be fully characterized.

[Podoplanin expression in oral squamous cell carcinoma and leukoplakia and its correlation with lymph vessels density].

OBJECTIVE: To investigate the expression of Podoplanin in lesional epithelia of oral leukoplakia and squamous cell carcinoma and its correlation with lymph vessels density. METHODS: The expression of Podoplanin in 40 cases of oral leukoplakia, 27 cases of early stage of squamous cell carcinoma and 32 cases of infiltrated stage of squamous cell carcinoma was examined by immunohistochemistry. Meanwhile, the correlation between podoplanin expression in lesional epithelia and its corresponding lymph vessels density was also analysed. RESULTS: Podoplanin expression in epithelia was increased with the exacerbated epithelial proliferation. High expression of Podoplanin in lesional epithelia accounted for 45% in oral leukoplakia, 81% and 78% in early and infiltrated stage of squamous cell carcinoma respectively. Moreover, the lymph vessels density was also increased with the enhanced expression of Podoplanin in oral leukoplakia and early stage of squamous cell carcinoma (P < 0.05). Podoplanin expression in infiltrated stage of squamous cell carcinoma was positively correlated with the lymph vessels density (P < 0.05). CONCLUSIONS: Podoplanin expression in different proliferative oral epithelia may be related to oral carcinogenesis, invasion and metastasis.