Clustering human dental pulp fibroblasts spontaneously activate NLRP3 and AIM2 inflammasomes and induce IL-1ß secretion.

Objectives: The objective of the present study was to investigate whether NOD-like receptor family pyrin domain-containing 3 (NLRP3) and absent in melanoma 2 (AIM2) inflammasomes pathways were involved in an experimental model of fibroblast activation named nemosis, which was used to mimic circumstances without bacteria stimulation. Methods: Nemosis of human dental pulp fibroblast (DPFs) was induced by three-dimensional culture in U-shaped 96-well plates and investigated by scanning electron microscopy (SEM). DPFs monolayers were used as control. Annexin V-FITC/7-AAD apoptosis assay was performed on the DPFs spheroids by flowcytometry. Caspase-1 activity detection assay was conducted on the DPFs spheroids. Quantitative real-time polymerase chain reaction (qRT-PCR), cytokine measurements, Western blot and the effect of COX-2 inhibitor on spheroids was studied. Results: SEM study observed human dental pulp fibroblast clusters and cell membranes damage on the surface of DPFs spheroids. The percentages of necrotic cells from DPFs spheroids gradually increased as the incubation time increased. A statistically significant increase in caspase-1 activity was observed after DPFs spheroids formation. DPFs spheroids displayed significant amounts of NLRP3, AIM2 mRNA and protein expression, caspase-1 mRNA expression and cleaved Caspase-1 protein expression and high IL-1ß concentrations (P < 0.05) than DPFs monolayers. Specific COX-2 inhibitor (NS-398) decreased NLRP3 mRNA and protein expression, cleaved Caspase-1 protein expression, Caspase-1 activity and IL-1ß mRNA expression and IL-1ß concentrations (P < 0.05). However, Specific COX-2 inhibitor had no impact on AIM2 mRNA and protein expression, caspase-1 mRNA expression and pro-Caspase-1 protein expression. Conclusions: In conclusion, clustering human DPFs spontaneously activated NLRP3 and AIM2 inflammasomes and induced IL-1ß secretion which could be partially attenuated by COX-2 inhibitor. Thus, nemosis could become a powerful model for studying mechanisms underlying aseptic pulpitis.

[Study on NaOH improving the surface morphology of three-dimensional printed poly- L- lactic acid mesh scaffolds].

Objective: To explore the effect of NaOH on the surface morphology of three-dimensional (3D) printed poly- L-lactic acid (PLLA) mesh scaffolds. Methods: The 3D printed PLLA mesh scaffolds were prepared by fused deposition molding technology, then the scaffold surfaces were etched with the NaOH solution. The concentrations of NaOH solution were 0.01, 0.1, 0.5, 1.0, and 3.0 mol/L, and the treatment time was 1, 3, 6, 9, and 12 hours, respectively. There were a total of 25 concentration and time combinations. After treatment, the microstructure, energy spectrum, roughness, hydrophilicity, compressive strength, as well as cell adhesion and proliferation of the scaffolds were observed. The untreated scaffolds were used as a normal control. Results: 3D printed PLLA mesh scaffolds were successfully prepared by using fused deposition molding technology. After NaOH etching treatment, a rough or micro porous structure was constructed on the surface of the scaffold, and with the increase of NaOH concentration and treatment time, the size and density of the pores increased. The characterization of the scaffolds by energy dispersive spectroscopy showed that the crystal contains two elements, Na and O. The surface roughness of NaOH treated scaffolds significantly increased ( P<0.05) and the contact angle significantly decreased ( P<0.05) compared to untreated scaffolds. There was no significant difference in compressive strength between the untreated scaffolds and treated scaffolds under conditions of 0.1 mol/L/12 h and 1.0 mol/L/3 h ( P>0.05), while the compression strength of the other treated scaffolds were significantly lower than that of the untreated scaffolds ( P<0.05). After co-culturing the cells with the scaffold, NaOH treatment resulted in an increase in the number of cells on the surface of the scaffold and the spreading area of individual cells, and more synapses extending from adherent cells. Conclusion: NaOH treatment is beneficial for increasing the surface hydrophilicity and cell adhesion of 3D printed PLLA mesh scaffolds.

Glucagon-like peptide-1 receptor promotes osteoblast differentiation of dental pulp stem cells and bone formation in a zebrafish scale regeneration model.

Bone cells express the glucagon-like peptide 1 receptor (GLP-1R). However, its presence and role in human dental pulp derived stem cells (hDPSCs) remains elusive. Hence, in the current study, we isolated hDPSCs and differentiated them into osteoblasts, where GLP-1R expression was found to be upregulated during osteoblast differentiation. GLP-1 receptor agonist, liraglutide peptide treatment, increased osteoblast differentiation in hDPSCs by increasing calcium deposition, ALP activity, and osteoblast marker genes, Runx2, type 1 col, osteonectin, and osteocalcin. Furthermore, activation of long non-coding RNA (LncRNA) LINC00968 and microRNA-3658 signalling increased Runx2 expression. Specifically, liraglutide increased LncRNA-LINC00968 expression while decreasing miR-3658 expression. LINC00968 targets miR-3658, and miR-3658 targets Runx2. Additionally, in an in-vivo study, zebrafish scale regeneration model, liraglutide promoted calcium deposition, osteoblastic cell count, collagen 1α, osteonectin, osteocalcin, runx2a MASNA isoform expression (transcribed from promoter P1), and Ca/P ratio in scales. Overall, GLP-1R activation promotes osteoblast differentiation via Runx2/LncRNA-LINC00968/miR-3658 signalling in hDPSCs and promotes bone formation in zebrafish scale regeneration.

[Applied anatomy of the stylomastoid foramina and its relation to the surrounding bone structures].

PURPOSE: To study the relationship between the stylomastoid foramen and surrounding bony structures, enrich anatomical data and provide reference for clinical surgery. METHODS: A total of 62 intact and dry adult skulls were selected. The shape of the stylomastoid foramen was observed, the diameter of the stylomastoid foramen, the distances from the posterolateral point and the anterior medial point to the surrounding bony structures were measured with a vernier caliper. SPSS 25.0 software package was used to analyze the data. RESULTS: There were four shapes of stylomastoid foramen, i.e., circular (61.29%), oval (29.84%), irregular (8.06%) and triangular (0.81%). The circular diameter was (2.80±0.61) mm, the oval long and short diameters were (4.43±0.96) and (2.79±0.60) mm. Distances from the posterolateral and anterior medial points of the stylomastoid foramen to the posterolateral point of the external opening of the carotid canal, the anterior medial point of the jugular foramen, the midline, the most anterior point of the foramen magnum, the posterior point of the great palatine foramen, the posterolateral point of the foramen lacerated, the foramen ovale, the posterolateral point of the foramen spinosum, the anterior point of the styloid process root, the outermost point of the tympanomastoid fissure and the tip of the mastoid process were (16.10±2.81), (24.01±2.65), (44.95±3.24), (45.10±2.71), (61.66±4.14), (35.56±4.35), (32.26±2.85), (29.12±3.40), (10.39±3.25), (9.49±2.24) and (12.01±2.79) mm; (12.80±2.41), (21.56±2.51), (42.96±3.97), (42.91±2.76), (58.97±3.97), (32.98±4.14), (29.20±2.77), (25.80±2.87), (7.37±2.33), (11.42±2.00) and (15.41±2.57) mm, respectively. Statistical analysis showed that there was no significant difference in the apertures and distances between the left and right side(P＞0.05). CONCLUSIONS: Most of the stylomastoid foramen are round and oval, understanding the distance between the foramen and surrounding bony structures is helpful for guiding clinical operations and enriching anatomical knowledge.

Immediate, non-submerged, three-dimensionally printed, one-piece mandibular molar porous root-analogue titanium implants: A 2-year prospective study involving 18 patients.

This study prospectively evaluated non-submerged, three-dimensionally printed, one-piece molar porous root-analogue titanium implants. A total of 18 non-restorable multiple-rooted teeth in 18 patients, aged 22-64 years, were included in this study. A series of computed tomography images of the mandible were selected and rendered into a digital model. The non-restorable mandibular molars were digitally separated from the surrounding alveolar bone, and served as the template on which the porous root-analogue titanium implants (RAIs) were designed with computer-aided design (CAD) software. The porous molar RAIs were fabricated with the selective laser melting technique (average particle size 20 µm) and inserted into the alveolar sockets after extraction of the non-restorable molars. Definitive restorations were placed after 3 months of uninterrupted healing. Peri-implant clinical and radiographic measurements were obtained 2 years later. All patients functioned well following 2 years of functional loading, and peri-implant clinical and radiographic measurements demonstrated implant stability. No implants were lost at the 2-year follow-up, and the survival rate was 100%. Three-dimensionally printed one-piece molar porous RAIs may be a promising option for the replacement of non-restorable molars that are planned for extraction. Additional studies are required to evaluate the long-term survival of implants fabricated using this technique.

An evaluation of allogeneic freeze-dried concentrated growth factors biocompatibilityin vitroandin vivo.

This study evaluated the biocompatibility of allogeneic freeze-dried concentrated growth factors (AFD-CGFs)in vitroandin vivo.For thein vitroexperiments, bone marrow stem cells (BMSCs) were cultured in 10% fresh allogeneic concentrated growth factors (CGFs). AFD-CGF solution was used as the experimental group, and Dulbecco's modified Eagle medium was used as control. Transmission electron microscopy (TEM) showed that the cell ultrastructure was unchanged, and membranes were intact. Scanning electron microscopy, cell counting kit-8, and quantitative polymerase chain reaction indicated that BMSCs and differentiation were unchanged between AFD-CGFs versus control groups (allp> 0.05). Alkaline phosphatase activity was higher in CGF groups (peaked at 14 d) than in the control group. Regarding thein vivoexperiments, four beagles were used for surgery and the rest as controls. Beagles were sacrificed at 2 weeks to observe acute response and membrane absorption; at 12 weeks for wound healing and chronic damage to the liver. According to general observations and histology, the CGFs of all groups were absorbed 2 weeks afterin vivoimplantation. No sign of intolerance was observed. Histology showed a slight increase in immune cells appearing in the implantation area after 2 weeks. However, no or very few inflammatory and immune cells were detected 3 months after the operation. Based on the hematoxylin and eosin staining and TEM results, the ultrastructure of the liver tissue was unchanged. In general, the results suggest that AFD-CGFs are biocompatible and may be a promising option for tissue healing.

Studies on the Performance of Molar Porous Root-Analogue Implant by Finite Element Model Simulation and Verification of a Case Report.

PURPOSE: The aim of this study was to evaluate the effect of porous layer thickness in a 3-dimensionally printed 1-piece molar porous root-analogue implant (RAI) on the biomechanical properties of the peri-implant bone and the clinical efficacy of one such implant in a patient. MATERIALS AND METHODS: Three RAIs with different superficial porous layer thicknesses (0.5 mm, 1 mm, and fully porous) were designed and assembled using a mandible model and then solidified to obtain 3 finite elements models, denoted A, B, and C. Finite element analysis was performed to analyze the stress on the solid and porous structures of the RAIs and the stress and strain experienced by the bone surrounding the implant. RAIs were fabricated by selective laser melting. An unrepairable molar in a single patient was selected for replacement. An RAI was designed and prepared and then implanted into the alveolar bone immediately after minimally invasive extraction of the damaged tooth. Definitive restorations were placed after a 3-month period of uninterrupted healing. RESULTS: The stress concentration observed in the 3 types of RAI was principally between the solid and porous interface contact points, with maximum stress on the solid and porous structures smaller than that of the respective yield strength. The introduction of a porous structure on the surface of the RAIs increased peri-implant bone stress, which increased with thickness of the porous layer. The 3-dimensionally printed porous RAI exhibited excellent initial stability immediately after implantation. After continual observation for 6 months, it was found that bone surrounding the root had infiltrated into the RAI, achieving good osseointegration. CONCLUSIONS: Stress shielding can be reduced by decreasing the elastic modulus of the implant, with the interface between implant and bone allowing more appropriate stress conduction. A 1-piece porous RAI fabricated using 3-dimensional printing establishes a new indication for immediate implantation after extraction.

Electron beam melting in the fabrication of three-dimensional mesh titanium mandibular prosthesis scaffold.

The study was designed to fulfill effective work-flow to fabricate three-dimensional mesh titanium scaffold for mandibular reconstruction. The 3D titanium mesh scaffold was designed based on a volunteer with whole mandible defect. (1) acquisition of the CT data; (2) design with computer aided design (CAD) and finite element analysis (FEA). The pore size and intervals with the best mechanic strength was also calculated using FEA. (3) fabrication of the scaffold using electron beam melting (EBM); (4) implantation surgery. The case recovered well, without loosening and rejection. Additionally, 12 mandibular defect model beagles were used to verify the results. The model was established via tooth extraction and mandibular resection surgeries, and the scaffold was designed individually based on CT data obtained at 2 weeks after extraction operation. Then scaffolds were fabricated using 3D EBM, and the implantation surgery was performed at 2 months after extraction operation. All the animals healed well after implantation, and the grafted mandibular recovered well with time. The relevant parameters of the grafted mandibular were nearly to the native mandibular at postoperative 12 months. It is feasible to fabricate mesh titanium scaffold for repairing mandibular defects individually using reverse engineering, CAD and EBM techniques.

Expression of long non-coding RNA-HOTAIR in oral squamous cell carcinoma Tca8113 cells and its associated biological behavior.

As a long noncoding RNA, HOX transcript antisense intergenic RNA (HOTAIR) is highly expressed in many types of tumors. However, its expression and function in oral squamous cell carcinoma (OSCC) cells and tissues remains largely unknown. We herein studied the biological functions of HOTAIR in OSCC Tca8113 cells. Real-time quantitative PCR showed that HOTAIR, p21 and p53 mRNA expressions in doxorubicin (DOX)-treated or γ-ray-irradiated Tca8113 cells were up-regulated. Knockdown of p53 expression inhibited DOX-induced HOTAIR up-regulation, suggesting that DNA damage-induced HOTAIR expression may be associated with p53. Transfection and CCK-8 assays showed that compared with the control group, overexpression of HOTAIR promoted the proliferation of Tca8113 cells, while interfering with its expression played an opposite role. Flow cytometry exhibited that HOTAIR overexpression decreased the rate of DOX-induced apoptosis. When HOTAIR expression was inhibited by siRNA, the proportions of cells in G2/M and S phases increased and decreased respectively. Meanwhile, the rate of DOX-induced apoptosis rose. DNA damage-induced HOTAIR expression facilitated the proliferation of Tca8113 cells and decreased their apoptosis. However, whether the up-regulation depends on p53 still needs in-depth studies.

Observational Study of Surgical Treatment of Sagittal Fractures of Mandibular Condyle.

OBJECTIVE: This study was conducted to investigate the complications that occur after surgical treatment of sagittal fracture of the mandibular condyle (SFMC). METHODS: A retrospective study was conducted on patients in whom SFMC was treated using surgical methods (87 patients, 105 sides) between January 1995 and December 2011 (79 sides were treated by rigid internal fixation and the remaining 26 sides were removed the condylar fragments). The longest follow-up was 17 years, and the shortest was 2 years. Follow-ups were conducted to assess mandibular activity, mouth opening, and computed tomography scans of condylar morphologic alterations. The postoperative complications were evaluated and the causes were analyzed. RESULTS: We observed 3 patients with joint ankylosis (all of them were removed the condylar fragments); 8, mouth opening less than 30 mm; 23, deviation on mouth opening at 6 months. At 4 weeks, 19 patients had facial nerve weakness, which was resolved within 6 months. The radiological investigation showed complete remodeling in 56.2% of the condyles (in the 59 sides, 57 sides were treated by rigid internal fixation and 2 sides were removed the condylar fragments); partial remodeling 27.6% condyles (in the 29 sides, 20 sides were treated by rigid internal fixation and 9 sides were removed the condylar fragments); poor remodeling, 16.2% condyles (in the 17 sides, 2 sides were treated by rigid internal fixation and 15 sides were removed the condylar fragments). CONCLUSIONS: Surgical treatment of SFMC is not perfect. There were some complications that occurred after the surgical treatment of SFMC. The findings also indicate that condylar anatomic reduction is the basis for functional recovery and, therefore, rigid fixation should be implemented. Furthermore, the removal of condylar fragments should be performed with caution, and if used, the fragments should be removed entirely.

[An experimental study on segmental defects reconstruction of canine mandible with allogenic bone marrow mesenchymal stem cells combined with lyophilized bone].

OBJECTIVE: To investigate the effect of segmental defects reconstruction of canine mandible with allogenenic bone marrow mesenchymal stem cells (BMSC) combined with lyophilized bone. METHODS: A 30 mm segmental defect was created on the left mandibles of beagles. Beagles were randomly divided into three groups. Allogeneic bone marrow mesenchymal stem cells with lyophilized bone were used for segmental defects reconstruction in group A. Autologous bone marrow mesenchymal stem cells with freeze- dried bone were used for segmental defects reconstruction in group B. The defects of group C were repaired with lyophilized bone only. Every three beagles were sacrificed 4, 12, 24, and 48 weeks after surgery respectively. The reconstruction effect was evaluated by CT and histopathological examination. RESULTS: CT examination showed that new bones formed in group A and group B 12 weeks after surgery, but not in group C. The form of the reconstructed mandibles in the three groups recovered in 48 weeks. The small pores on the bone graft were filled with new bones in group A and group B. In group C, the pores were still evident. Histopathological examination showed that bone trabecula between allogeneic bone and autogenous bone was completely joined in group A and group B. A large number of fibrous tissue appeared around the implanted bone and new bones were formed. In group C, the lyophilized bone resorption was still not obvious, the new bone formation was significantly slower than the other two groups. There was no difference between group A and group B. CONCLUSIONS: Both allogeneic bone marrow mesenchymal stem cells and autologous mesenchymal stem cells could accelerate the bone formation.

[Clinical analysis of 12 cases of orthognathic surgery with digital computer-assisted technique].

PURPOSE: This study was to investigate the effect of the digital computer-assisted technique in orthognathic surgery. METHODS: Twelve patients from January 2008 to December 2011 with jaw malformation were treated in our department. With the help of CT and three-dimensional reconstruction technique, 12 patients underwent surgical treatment and the results were evaluated after surgery. RESULTS: Digital computer-assisted technique could clearly show the status of the jaw deformity and assist virtual surgery. After surgery all patients were satisfied with the results. CONCLUSIONS: Digital orthognathic surgery can improve the predictability of the surgical procedure, and to facilitate patients' communication, shorten operative time, and reduce patients' pain.

Reconstruction of beagle hemi-mandibular defects with allogenic mandibular scaffolds and autologous mesenchymal stem cells.

OBJECTIVE: Massive bone allografts are frequently used in orthopedic reconstructive surgery, but carry a high failure rate of approximately 25%. We tested whether treatment of graft with mesenchymal stem cells (MSCs) can increase the integration of massive allografts (hemi-mandible) in a large animal model. METHODS: Thirty beagle dogs received surgical left-sided hemi-mandibular defects, and then divided into two equal groups. Bony defects of the control group were reconstructed using allografts only. Those of the experimental group were reconstructed using allogenic mandibular scaffold-loaded autologous MSCs. Beagles from each group were killed at 4 (n = 4), 12 (n = 4), 24 (n = 4) or 48 weeks (n = 3) postoperatively. CT and micro-CT scans, histological analyses and the bone mineral density (BMD) of transplants were used to evaluate defect reconstruction outcomes. RESULTS: Gross and CT examinations showed that the autologous bone grafts had healed in both groups. At 48 weeks, the allogenic mandibular scaffolds of the experimental group had been completely replaced by new bone, which has a smaller surface area to that of the original allogenic scaffold, whereas the scaffold in control dogs remained the same size as the original allogenic scaffold throughout. At 12 weeks, the BMD of the experimental group was significantly higher than the control group (p<0.05), and all micro-architectural parameters were significantly different between groups (p<0.05). Histological analyses showed almost all transplanted allogeneic bone was replaced by new bone, principally fibrous ossification, in the experimental group, which differed from the control group where little new bone formed. CONCLUSIONS: Our study demonstrated the feasibility of MSC-loaded allogenic mandibular scaffolds for the reconstruction of hemi-mandibular defects. Further studies are needed to test whether these results can be surpassed by the use of allogenic mandibular scaffolds loaded with a combination of MSCs and osteoinductive growth factors.

Recurrent Pleomorphic Adenoma of the Parotid Gland: Intraoperative Facial Nerve Monitoring during Parotidectomy.

OBJECTIVE: To determine the benefit to postoperative facial function of intraoperative facial nerve monitoring (IFNM) during recurrent pleomorphic adenoma (RPA) parotid surgery. STUDY DESIGN: Cohort study with planned data collection. SETTING: Chinese PLA General Hospital. SUBJECTS AND METHODS: Fifty-eight RPA parotidectomies were performed between 2004 and 2012. Recurrence was confirmed by histopathologic examination. None of the patients had preoperative facial palsy. Electromyography-based IFNM was used in 28 patients; 30 patients were unmonitored. The durations of surgery and the severity of postoperative facial nerve palsy were compared between the 2 groups. RESULTS: There were no significant differences between the 2 groups in the incidence of immediate or permanent facial paralysis after RPA parotidectomy (P = .95 and P = .36, respectively). However, the differences in the average duration of surgery and the severity of postoperative facial nerve palsy after total parotidectomy or wide resection were significant (P < .01 and P = .01, respectively). In contrast, these differences were not significant after superficial parotidectomies (P = .43 and P = .49, respectively). The average recovery time of temporary facial nerve paralysis was significantly shorter in the monitored group compared with the unmonitored group, independent of surgical technique (P < .01). CONCLUSION: The use of IFNM during total or wide resection RPA parotidectomy reduced the duration of surgery and the incidence of postoperative facial paralysis and enhanced recovery. However, there was little impact on facial nerve outcomes when IFNM was used during superficial RPA parotidectomy.

Clinical and radiological outcomes after treatment of sagittal fracture of mandibular condyle (SFMC) by using occlusal splint in children.

This study was designed to investigate the effects of occlusal splints in the treatment of sagittal fractures of the mandibular condyle in children. From January 1995 to December 2011, 37 sagittal fractures of the mandibular condyle in 30 patients aged 4-8 years old were included in this study. All the patients were treated with 1-2mm occlusal splints in the molar region. The mouths of the patients were kept slightly open by the occlusal splints for 3-6 months, and we reviewed the clinical and radiological remodelling of the affected condyles after treatment. Excellent (n=20) and good (n=10) clinical outcomes were achieved with full radiological remodelling seen in 19 and partial remodelling in 11. Treatment with occlusal splints is effective in delivering good results and function with minimal morbidity in children with sagittal fractures of the condyle, while permitting ongoing remodelling and growth in the short term.