Aspirin/amoxicillin loaded chitosan microparticles and polydopamine modified titanium implants to combat infections and promote osteogenesis.

It is known that titanium (Ti) implant surfaces exhibit poor antibacterial properties and osteogenesis. In this study, chitosan particles loaded with aspirin, amoxicillin or aspirin + amoxicillin were synthesized and coated onto implant surfaces. In addition to analysing the surface characteristics of the modified Ti surfaces, the effects of the modified Ti surfaces on the adhesion and viability of rat bone marrow-derived stem cells (rBMSCs) were evaluated. The metabolic activities of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) biofilms on the modified Ti surfaces were also measured in vitro. Moreover, S. aureus was tested for its antibacterial effect by coating it in vivo. Using water as the droplet medium, the contact angles of the modified Ti surfaces increased from 44.12 ± 1.75° to 58.37 ± 4.15°. In comparison to those of the other groups tested, significant increases in rBMSC adhesion and proliferation were observed in the presence of aspirin + amoxicillin-loaded microspheres, whereas a significant reduction in the metabolic level of biofilms was observed in the presence of aspirin + amoxicillin-loaded microspheres both in vitro and in vivo. Aspirin and amoxicillin could be used in combination to coat implant surfaces to mitigate bacterial activities and promote osteogenesis.

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.

A Comparative Evaluation of Iliac Crest Cortical-Cancellous Bone Blocks Graft With and Without Concentrated Growth Factors (CGFs) in Secondary Alveolar Bone Grafting: A Retrospective Study.

BACKGROUND: The purpose of this study was to investigate the clinical effect and bone resorption of iliac crest cortical-cancellous bone block grafts combined with concentrated growth factor (CGF) compared with iliac crest cortical-cancellous bone block grafts only in secondary alveolar bone grafting. MATERIALS AND METHODS: Eighty-six patients (43 in the CGF group and 43 in the non-CGF group) with unilateral alveolar clefts were examined. Patients (17 in the CGF group and 17 in the non-CGF group) were randomly chosen for radiologic evaluation. Quantitative evaluation of the bone resorption rate was made with cone-beam computed tomography and Mimics 19.0 software at 1 week and 12 months after surgery. RESULTS: The success rate of bone grafting was 95.3% and 79.1% in the CGF and non-CGF groups, respectively ( P =0.025). The mean bone resorption rate at 12 months postoperatively was 35.66±15.80% and 41.39±19.57% in the CGF and non-CGF groups, respectively ( P =0.355). The bone resorption patterns of the 2 groups were similar on the labial, alveolar process, and palatal sides, and there was no obvious bone resorption on the labial side in either group. Nasal side bone resorption in the CGF group was significantly less than that in the non-CGF group ( P =0.047). CONCLUSIONS: Cortical-cancellous bone block grafts reduce labial bone resorption, while CGF reduces nasal bone resorption and improves the success rate. The combination of bone block and CGF in secondary alveolar bone grafting is worthy of further clinical application.

Mandible reconstruction with free fibula flaps: Accuracy of a cost-effective modified semicomputer-assisted surgery compared with computer-assisted surgery - A retrospective study.

A new individualized, cost-effective, modified semi-computer-assisted surgery (MSCAS) concept for free fibular flap mandibular reconstruction is reported and compared with the computer-assisted surgery (CAS) concept. Patients were divided into two groups and retrospectively reviewed. In the MSCAS and CAS groups, intraoperative guides were created using computer-aided design with manual fabrication and computer-aided design and manufacturing, respectively. Differences in specific linear and angular parameters on pre- and postoperative computed tomography scans were calculated for morphometric comparison, and clinical parameters and efficiency were analysed. RESULTS: Eighteen patients (CAS, 7; MSCAS, 11), were included. The morphometric comparison showed no significant differences between the groups. The mean deviation of the mandibular ramus length, body length, width 1 and width 2 was 0.82 ± 0.29 mm, 1.84 ± 0.43 mm, 1.89 ± 0.61 mm and 1.45 ± 0.61 mm in the CAS group versus 1.56 ± 0.54 mm, 1.72 ± 0.33 mm, 2.24 ± 0.55 mm and 2.36 ± 0.50 mm in the MSCAS group (p = 0.7804, p = 0.9997, p = 0.9814 and p = 0.6334). The mean deviation of the sagittal, axial and coronal mandibular angles was 1.56 ± 0.48°, 1.93 ± 0.50° and 2.15 ± 0.72° in the CAS group versus 2.19 ± 0.35°, 1.86 ± 0.35° and 1.94 ± 0.55° in the MSCAS group (p = 0.7594, p = 0.9996 and p = 0.9871). There were no significant differences in clinical parameters, efficiency or postoperative complications between the groups. CONCLUSION: The accuracy and operative efficiency of the MSCAS concept are comparable to those of the more expensive CAS concept. Therefore, in times of increasing clinical costs, this concept might be an adequate and inexpensive alternative to preoperative CAS.

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.

Preliminary efficacy analysis of block iliac bone grafting in patients with alveolar cleft in mixed dentition.

OBJECTIVES: To investigate the efficacy of block iliac bone grafting in patients with unilateral alveolar cleft in mixed dentition. METHODS: A retrospective study was conducted on patients with unilateral alveolar clefts in mixed dentition who were treated in the Department of Oral and Maxillofacial Surgery, Fujian Medical University Union Hospital. All patients underwent unilateral alveolar cleft bone graft repair with autogenous block iliac bone blocks. The healing of bone blocks was analyzed at 1 week and 6-12 months after surgery. Mimics software was used for the three-dimensional reconstruction and volumetric measurement of the iliac bone blocks on the follow-up imaging data of 15 patients aged 9-12 years without the eruption of canines before surgery, and the bone resorption rate of the iliac bone blocks was comparatively analyzed. RESULTS: In the 37 patients, bone grafting was successful in 32 and failed in five. The success rate of bone grafting was 86.5%. In 15 patients aged 9-12 years without the eruption of canines before surgery, eruption through the bone graft area was observed in two patients 6-12 months after the operation. Cone beam computer tomography showed that the grafted bone block exhibited good bony connections, and its resorption mainly occurred on the crests and palatal sides of the alveolar ridge. Bone resorption rates varied considerably between patients with a mean bone resorption rate of 39.0%±13.8% at 6-12 months after surgery. CONCLUSIONS: For patients in mixed dentition, bone grafting with block iliac bone can achieve better osteogenesis effect.

MMP1 3'UTR facilitates the proliferation and migration of human oral squamous cell carcinoma by sponging miR-188-5p to up-regulate SOX4 and CDK4.

Growing evidence indicates that the non-coding 3'-untranslated region (3'UTR) of genes acts as competing endogenous RNAs (ceRNAs) to exert their roles in a number of diseases, including cancer. In the present study, MMP1 messenger RNA was identified to be significantly up-regulated in oral squamous cell carcinoma (OSCC) tissues, and both MMP1 and its 3'UTR promoted tumor growth and cell motility. Further mechanism investigations indicated that MMP1 3'UTR was able to antagonize miR-188-5p; in addition, overexpression of MMP1 3'UTR up-regulated the expression level of SOX4 and CDK4, target genes of miR-188-5p, which have also been identified as oncogenic driver genes in OSCC. Therefore, a ceRNA regulatory network among MMP1, SOX4, and CDK4 mediated via competing for binding to miR-188-5p was proved. Taken together, the present study demonstrates for the first time that MMP1 mRNA participates in the development of OSCC via ceRNA regulatory mechanism and genes involved in the ceRNA network may provide a novel avenue for target therapy.

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.

The preventive effect of decellularized pericardial patch against Frey's syndrome following the superficial parotidectomy.

PURPOSE: This study aimed to investigate the feasibility of adopting the ThormalGEN surgical patch (a porcine decellularized pericardial patch) in the superficial parotidectomy to prevent postoperative Frey's syndrome in patients with benign parotid tumor. MATERIALS AND METHODS: A total of 59 patients with benign parotid tumor undergoing superficial parotidectomy between April 2016 and January 2017 were enrolled, and divided into the ThormalGEN group (n = 37) and the control group (n = 22) based on their willingness to have the ThormalGEN surgical patch used in the superficial parotidectomy. At 6 months postoperation, the incidences of postoperative complications and Frey's syndrome were assessed by subjective assessment (patient self-assessment) and objective assessment (starch-iodine test), respectively. The risk factor of Frey's syndrome was analyzed by the multivariate logistic regression model. RESULTS: The starch-iodine test, for objective assessment, showed that the ThormalGEN group had a significantly lower incidence of Frey's syndrome than the control group (8.11% vs. 40.91%, p = 006). However, in the patient self-assessment, there was no significant difference in the incidence of Frey's syndrome between groups (8.11% [3/37] vs. 13.64% [3/22], p = 0.4968). The incidences of postoperative complications were not significantly different between group (all p > 0.05). Two patients (5.41%) in the ThormalGEN group had salivary fistula. Multivariate logistic regression analysis showed that the ThormalGEN group had a significantly lower risk of Frey's syndrome than the control group (odds ratio = 0.11, 95% confidence interval = 0.02-0.51, p < 0.01). CONCLUSION: These results suggest that the ThormalGEN surgical patch can effectively reduce the incidence of Frey's syndrome following superficial parotidectomy in patients with benign parotid tumor, and that this patch has a good safety and biocompatibility profile.

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.

miR-654-5p Targets GRAP to Promote Proliferation, Metastasis, and Chemoresistance of Oral Squamous Cell Carcinoma Through Ras/MAPK Signaling.

Oral squamous cell carcinoma (OSCC) is characterized by rapid local migration and invasion. This study was aimed at clarifying the effect of miR-654-5p on progression of OSCC. miR-654-5p promoted proliferation, metastasis, and chemoresistance of OSCC in vitro and in vivo. Consistently, miR-654-5p was upregulated in late-stage OSCC and was correlated with poor prognosis of OSCC patients. Furthermore, miR-654-5p was mechanistically verified to target Grb-2-related adaptor protein (GRAP), accompanied by the activation of Ras/MAPK signaling and the facilitation of epithelial-mesenchymal transition in OSCC cells. GRAP was downregulated in T1-2 stage versus T3-4 stage head and neck squamous cell carcinoma (HNSC) and was negatively correlated with tumor-node-metastases (TNM) stage in HNSC patients based on The Cancer Genome Atlas (TCGA) analysis. In addition, GRAP was positively correlated with good prognosis in HNSC patients. Our findings suggest that the miR-654-5p/GRAP/Ras/Erk signaling pathway in OSCC cells might contribute to the underlying mechanism through which miR-654-5p participates in the regulation of OSCC progression. miR-654-5p, as a potential biomarker for the clinical diagnosis and prognosis of OSCC, may be an effective anticancer target for the treatment of OSCC.

Reciprocal regulation of miR-1254 and c-Myc in oral squamous cell carcinoma suppresses EMT-mediated metastasis and tumor-initiating properties through MAPK signaling.

AIM: This study aimed to determine the effect of miR-1254 on oral squamous cell carcinoma (OSCC) metastasis and the specific mechanism involved. METHODS: The metastatic properties of OSCC cells were analyzed by transwell assays. The tumor-initiating properties of OSCC cells were analyzed by tumor sphere formation assays. The mRNA and protein expressions of targeted genes were determined by quantitative polymerase chain reaction assays and western blot analyses, respectively. Xenograft experiments were employed to evaluate the anti-metastatic effects of miR-1254 and miR-1254-mediated cancer stem cell (CSC) properties in vivo. The gene targets of miR-1254 were investigated by luciferase reporter assays. Chromatin immunoprecipitation assays were performed to observe the transcriptional regulation of miR-1254 biogenesis by transcription factor. RESULTS: miR-1254 attenuated OSCC metastasis and tumor-initiating properties in vitro and in vivo. Consistent with the experimental observations, miR-1254 was decreased in late-stage OSCCs and strongly correlated with risk of OSCC metastasis. Moreover, miR-1254 was mechanistically shown to down-regulate MAP3K3, accompanied by inactivation of the MAPK signaling pathway and inhibition of epithelial-mesenchymal transition (EMT) in OSCC cells. miR-1254 was transcriptionally repressed by c-Myc to form a positive feed back loop through MAPK signaling. CONCLUSION: Our findings suggest that miR-1254 is a potential target for the treatment of OSCCs, and miR-1254 can be clinically utilized as a biomarker for the clinical prognosis or diagnosis of OSCCs.

Beta-nerve growth factor promotes neurogenesis and angiogenesis during the repair of bone defects.

We previously showed that the repair of bone defects is regulated by neural and vascular signals. In the present study, we examined the effect of topically applied ß-nerve growth factor (ß-NGF) on neurogenesis and angiogenesis in critical-sized bone defects filled with collagen bone substitute. We created two symmetrical defects, 2.5 mm in diameter, on either side of the parietal bone of the skull, and filled them with bone substitute. Subcutaneously implanted osmotic pumps were used to infuse 10 µg ß-NGF in PBS (ß-NGF + PBS) into the right-hand side defect, and PBS into the left (control) defect, over the 7 days following surgery. Immunohistochemical staining and hematoxylin-eosin staining were carried out at 3, 7, 14, 21 and 28 days postoperatively. On day 7, expression of ß III-tubulin was lower on the ß-NGF + PBS side than on the control side, and that of neurofilament 160 was greater. On day 14, ß III-tubulin and protein gene product 9.5 were greater on the ß-NGF + PBS side than on the control side. Vascular endothelial growth factor expression was greater on the experimental side than the control side at 7 days, and vascular endothelial growth factor receptor 2 expression was elevated on days 14 and 21, but lower than control levels on day 28. However, no difference in the number of blood vessels was observed between sides. Our results indicate that topical application of ß-NGF promoted neurogenesis, and may modulate angiogenesis by promoting nerve regeneration in collagen bone substitute-filled defects.