Saliva biopsy: Detecting the difference of EBV DNA methylation in the diagnosis of nasopharyngeal carcinoma.

Saliva sampling is a non-invasive method, and could be performed by donors themselves. However, there are few studies reporting biomarkers in saliva in the diagnosis of NPC. A total of 987 salivary samples were used in this study. First, EBV DNA methylation was profiled by capture sequencing in the discovery cohort (n = 36). Second, a q-PCR based method was developed and five representative EBV DNA CpG sites (11 029 bp, 45 849 bp, 57 945 bp, 66 226 bp and 128 102 bp) were selected and quantified to obtain the methylated density in the validation cohort1 (n = 801). Third, a validation cohort2 (n = 108) was used to further verify the differences of EBV methylation in saliva. A significant increase of EBV methylation was found in NPC patients compared with controls. The methylated score of EBV genome obtained by capture sequencing could distinguish NPC from controls (sensitivity 90%, specificity 100%). Further, the methylated density of EBV DNA CpG sites revealed by q-PCR showed a good diagnostic performance. The sensitivity and specificity of detecting a single CpG site (11 029 bp) could reach 75.4% and 99.7% in the validation cohort1, and 78.2% and 100% in the validation cohort2. Besides, the methylated density of the CpG site was found to decrease below the COV in NPC patients after therapy, and increase above the COV after recurrence. Our study provides an appealing alternative for the non-invasive detection of NPC without clinical setting. It paves the way for conducting a home-based large-scale screening in the future.

The Oral Microbiome as Mediator between Oral Hygiene and Its Impact on Nasopharyngeal Carcinoma.

Oral hygiene and the alteration of the oral microbiome have been linked to nasopharyngeal carcinoma (NPC). This study aimed to investigate whether the oral microbiome plays a mediating role in the relationship between oral hygiene and NPC, and identify differential microbial taxonomies that potentially mediated this association. We conducted a case-control study that involved 218 NPC patients and 192 healthy controls. The 16S rRNA gene sequencing of the V4 region was performed to evaluate the composition of the oral microbiome. Mediation analysis was applied to explore the relationship among oral hygiene, the oral microbiome and NPC. We found that dental fillings and poor oral hygiene score were associated with increased risks of NPC (OR = 2.51 (1.52-4.25) and OR = 1.54 (1.02-2.33)). Mediation analysis indicated that dental fillings increased the risk of NPC by altering the abundance of Erysipelotrichales, Erysipelotrichaceae, Solobacterium and Leptotrichia wadei. In addition, Leptotrichia wadei also mediated the association between oral hygiene score and the risk of NPC. Our study confirmed that poor oral hygiene increased the risk of NPC, which was partly mediated by the oral microbiome. These findings might help us to understand the potential mechanism of oral hygiene influencing the risk of NPC via the microbiome.

[Odontoblast-like cell phenotype differentiation of cranial neural crest stem cell in vivo].

OBJECTIVE: To investigate the feasibility of tooth regeneration by seeding cranial neural crest stem cell (CNCSC) in vivo. METHODS: Cranial neural tubes, dissected from mouse E9 d, were explanted onto fibronectin-coated dishes. CNCSC emigrated from the explanted neural tubes, and were cultured in a free-serum medium containing modified DMEM/F12. CNCSC, induced by FGF8, BMP2, TGFbeta1 and dentin matrix non-collagen protein (DMNCP), were cultured with collagen/chitosan, and implanted into the subcutaneous part of immunodeficiency mouse. The expression of collagen I/dentin sialophosphoprotein (DSPP) was analyzed by immunocytochemistry. RESULTS: With the scaffolds destroying, columnar cells possessing polarized nuclei and matrix produced by cells were showed in some regions. Immunohistochemical staining demonstrated that collagen type I and DSPP were expressed throughout the cytoplasm and matrix produced by cells. CONCLUSION: By tissue engineering approach, our experiments further verify the odontoblast-like cell phenotype differentiation of CNCSC in vivo.

[Construction of recombinant adenovirus vector carrying the mouse caveolin-1 in gene].

OBJECTIVE: To construct recombinant adenovirus carrying the mouse dentin caveolin-1 gene using the recombinant adenoviral vector system AdEasy. METHODS: The cDNA fragment of caveolin-1 was derived from pTRE2-caveolin-1 by restriction enzyme digestion and subcloned into shuttle plasmid pAdtrack-CMV. The resulting plasmid pAdtrack-CMV-caveolin-1, after linearized by digesting with restriction endonuclease Pme I, was transformed into E. coli 1 BJ5183 which had been transformed by adenoviral backbone plasmid pAdEasy-1. The recombinant plasmid pAd-caveolin-1 was screened by kanamycin LB plate and then identified by restriction enzyme digestion. The linearized adenovirus plasmid pAd-caveolin-1 was packaged in 293 cells, then the recombinant adenovirus Ad-caveolin-1 was harvested. The expression of green fluorescence protein was observed under fluorescent microscope. With further amplification and purification, the titer of recombinant adenovirus was determined. RESULTS: The recombinant adenovirus was identified by restriction enzyme digestion analysis and gene sequencing. Cytopathic effect and the expression of GFP were observed in the infected 293 cells. The sequence of caveolin-1 gene insert was the same as that published in the GenBank. The titer of the recombinant adenovirus was 2 x 10(9) pfu/mL. CONCLUSION: The mouse caveolin-1 recombinant adenovirus was constructed successfully by using AdEasy adenovirus system. Cell transfection and expression of exogenous gene can be detected directly by observing the expression of GFP. These results provide the basis for the further study on the role of caveolin-1 gene in other scopes.

[Mandibular distraction osteogenesis: an experimental study in goats].

OBJECTIVE: This experiment was designed to develop an experimental model of mandibular distraction osteogenesis in goats for determining the principle and the process of distraction osteogenesis. METHODS: Eight 3-month-old, healthy domestic goats were randomly divided into four groups (n = 2 per group). The animals from each group were killed at 1, 2, 4, and 6 weeks after bone distraction, respectively. The sections of newly formed bone were obtained and evaluated with histologic and radiographic study. We also assessed the newly formed bone with bone density examination. RESULTS: The specimens of right mandible increased averagely 10 mm after distraction. The Newly formed bone was demonstrated histologically. The X-ray examination and bone density measurements showed that the bone density increased with the time of fixation. CONCLUSION: Goat is a proper experimental animal for modeling mandibular distraction osteogenesis, and in this regal, it is important to keep the direction of distracter force in conformity with the direction of the expected distraction osteogenesis for avoiding the side force and thus ensuring successful distraction and good quality of newly formed bone.

New bone formation enhanced by ADSCs overexpressing hRunx2 during mandibular distraction osteogenesis in osteoporotic rabbits.

Promoting new bone formation during distraction osteogenesis (DO) in elderly patients with osteoporosis is still a challenge. In this study, we investigated the effect of gene therapy using local Runt-related gene 2 on new bone formation during osteoporotic mandibular DO in rabbits. First, we successfully established a mandibular osteoporotic animal model by ovariectomizing rabbits. Second, the right mandibles of the osteoporotic rabbits were distracted after corticotomy. The distraction gap of the rabbits in Group A2 and B2 were injected with Adv-hRunx2-GFP-transfected adipose-derived stromal cells (ADSCs) and Adv-GFP-transfected ADSCs, respectively. Rabbits in Groups C2 (ovariectomized control) and D2 (sham surgery control) were injected with physiologic saline. New-generation bone tissue in the distraction gap was analyzed via plain radiographic examinations, micro-computed tomography, histological examinations, and biomechanical testing at weeks 3, 6, and 9 of the consolidation period. Results of above examinations showed that no ideal new bone formation was observed in Groups B2 and C2, but obvious ideal new bone formation was observed in Group A2 and D2. The results suggested that gene therapy using rhRunx2-modified ADSCs promoted new bone formation during osteoporotic mandibular DO and effectively compensated for the detrimental effects of systemic osteoporosis on new bone formation.

Effects of bone morphogenetic protein 2 gene therapy on new bone formation during mandibular distraction osteogenesis at rapid rate in rabbits.

OBJECTIVE: We investigated the effect of recombinant human bone morphogenetic protein 2 (rhBMP-2) on new bone formation during rapid-rate mandibular distraction osteogenesis. We also explored the feasibility of using local BMP-2 gene therapy to compensate for bad callus formation caused by a rapid distraction rate. STUDY DESIGN: Bone marrow mesenchymal stem cells (MSCs) from Japanese rabbits were transfected with adenovirus (adv)-BMP-2. The right mandibles of the rabbits were distracted after corticotomy. The distraction rate in group A was 0.8 mm/d. The distraction rate in group B was 2.4 mm/d, and the distraction gap was injected with adv-lacZ-transfected bone marrow MSCs. The distraction rate in group C was 2.4 mm/d, and the distraction gap was injected with adv-BMP-2-transfected bone marrow MSCs. New generation bone tissue in the distraction gap was analyzed by plain radiograph examinations, microfocus computerized tomography (micro-CT) examinations, and biomechanical tests at weeks 2, 4, and 8 of the consolidation period. RESULTS: Radiographic and micro-CT examinations showed a better bone quality in group C compared with group A at weeks 2 and 4 of the consolidation period. There was no obvious new bone formation in group B. The trabecular parameters (trabecular thickness, trabecular number, volumetric bone mineral density at tissue, and bone volume fraction) were significantly higher in group C than in group A at weeks 2 and 4. At week 8, no significant difference were detected for all parameters except trabecular number between groups A and C. All biomechanical stress parameters were significantly higher in group C than in group A at week 4, and only peak stress was significantly different at week 8. CONCLUSIONS: Gene therapy using rhBMP-2-modified MSCs promoted new bone formation during mandibular distraction osteogenesis, and effectively compensated for the detrimental effect of rapid distraction rate on new bone formation.

Effects of rapid distraction rate on new bone formation during mandibular distraction osteogenesis in goats.

OBJECTIVE: Distraction osteogenesis typically requires a long treatment period, which can lead to bone and soft-tissue infection and considerable patient discomfort. Use of a rapid distraction rate in craniofacial distraction osteogenesis to shorten the distraction period is possible owing to the unique characteristics of craniofacial bones, including an abundant blood supply and rapid bone healing compared with long bones. The effects of using a rapid distraction rate in the treatment of craniofacial deformities are currently unclear, however. The objective of this study was to investigate the effects of a rapid distraction rate on new bone formation during mandibular distraction osteogenesis in goats. METHODS: Sixteen goats were randomly divided into four groups consisting of four goats each. In Groups A, B, and C, the right mandible of each goat was distracted at a rate of 0.8mm/d, 1.6mm/d, and 2.0mm/d, respectively; Group D was the control group and did not undergo distraction. Six weeks after the conclusion of distraction, bone densitometry and three-point bending testing were performed in all groups. RESULTS: The mean bone density value of goats in Group A was significantly higher than those of all the other groups (p<0.05), and the mean bone density value of goats in Group C was significantly lower than those of all the other groups (p<0.05). The mean curve slope, peak stress, bending modulus, and energy to failure values of Groups A, B, and C were all significantly lower than those of the control group (p<0.05). As the distraction rate increased, the curve slope and peak stress values gradually declined (p<0.05). CONCLUSIONS: Use of a rapid distraction rate in mandibular distraction osteogenesis may have detrimental effects on the quality of new bone, despite the abundant blood supply of craniofacial bones.

[Clinical analysis of midfacial fractures with orbital floor fractures].

OBJECTIVE: To study the characters of the diagnosis and treatment for midfacial fractures with orbital floor fractures. METHODS: 136 cases of midfacial fractures with orbital floor fractures were diagnosed and treated. All patients underwent CT scans and accepted surgical intervention. Orbital floor fracture was restituted in 49 cases. Orbital floor defects were reconstructed with autogenous bone, titanium mesh or porous polyethylene implant (Medpor) in 21 cases. RESULTS: 136 cases recovered well, their facial appearance and function were improved significantly. There were no severe complications happened postoperatively in all cases. 2 cases had postoperative wounds infection and 1 case had temporary ablepsia, but healed completely. CONCLUSION: CT is the best method for diagnosing orbital floor fractures. The objective of treatment is restoration of normal orbital floor and orbital capacity, the reduction of orbital contents prolapsed and reconstruction of orbital floor fractures with repair materials.

[Culture and characteristics of human dental papilla cells in vitro].

OBJECTIVE: To culture human dental papilla cells (HDPCs)and to study its cytobiological characters in vitro. METHODS: HDPCs were isolated and cultured with explant culture technique in vitro; Type I collagen, fibronection and laminin were detected in HDPCs and its secreted matrix with the immunocyto-chemical stain; HDPCs were incubated in mineralized promoting solution containing 10 mmol/L beta-glycerophosphate, 100 mg/L of ascorbic acid and 10 nmol/L dexamethasone supplemented with 10% FBS and the form of mineralized nodules was tested with Alizarin Red S stainning. RESULTS: Cultured HDPCs in vitro were well growing in DMEM/F12. Type I collagen, fibronection and laminin staining were all positive in both HDPCs and its secreted matrix, and laminin was stained with bunchiness in matrix. Mineralized nodules formed after cultured 27 days by Alizarin Red S stainning. CONCLUSION: HDPCs isolated and cultured are well growing in vitro, have a capability of synthesizing and secreting matrix and in mineralized promoting solution, are able to form mineralizer, so, HDPCs have a capacity of seed cell of tissue engineering regeneration tooth.