Peri-tumoral infiltrate associates with postoperative prognosis of patients with OSCC: Stronger association in HPV negative patients.

PURPOSE: The current data on the relationship between local inflammatory infiltration and prognosis in oral squamous cell carcinoma (OSCC) are limited and controversial, especially in different HPV status. In this study, we analyzed the relationship between peri-tumoral inflammatory infiltrate (PTI) and HPV status and prognosis of patients with OSCC after surgery. METHODS: A retrospective cohort of 99 primary OSCC patients who underwent surgery was constructed. P16 immunohistochemistry was used to determine HPV status. PTI was determined by hematoxylin-eosin staining and quantified into four levels: none (Score 0), weak (Score 1), moderate (Score 2) and strong (Score 3). The associations of PTI with clinico-pathological characteristics, HPV status and survival were examined. RESULTS: Most OSCC patients had weak to moderate PTI. PTI was significantly associated with lymph node metastasis (P = 0.041), and patients with moderate PTI had significantly better OS (P = 0.009) than those with no PTI. In HPV negative OSCC, patients with moderate PTI also had significantly better OS (P = 0.019) than those with no PTI. However, PTI was not significantly associated with survival in HPV positive OSCC. CONCLUSIONS: In HPV negative OSCC, moderate PTI may suggest a better postoperative prognosis than no PTI.

Identification and characterization of circular RNAs involved in mechanical force-induced periodontal ligament stem cells.

Circular RNAs (circRNAs) play critical roles in signal transduction during cell proliferation, differentiation, and apoptosis in a posttranscriptional manner. Recently, circRNAs have been proved to be a large class of animal RNAs with regulatory potency. However, whether circRNAs can respond to mechanical force (MF) and impact on human periodontal ligament stem cells (PDLSCs) and the orthodontic tooth movement (OTM) process remain unknown. Here, we investigated the circRNAs expression patterns in PDLSCs induced by MF and found that circRNAs were responsive to the MF in PDLSCs. Through the valid reads' distribution analysis, we found that the majority of reads in both the control PDLSCs and the MF-induced PDLSCs were distributed in exons. Then we analyzed Gene Ontology terms of genes that overlap with or are neighbors of the stress-responsive circRNAs and found unique enrichment patterns in biological processes, molecular function, and cellular component of PDLSCs. Next, we predicted the possible functions of circRNAs through circRNAs-miRNAs networks. We found that one circRNA may regulate one or several miRNA/miRNAs and one miRNA may interact with one or multiple circRNA/circRNAs. Importantly, a number of circRNAs were predicted to directly or indirectly regulate miRNAs-mediated osteogenic differentiation in mesenchymal stem cells. For instance, circRNA3140 was highly and widely associated with microRNA-21, which plays a critical role in MF-induced osteogenic differentiation of PDLSCs. Taken together, these findings reveal a previously unrecognized mechanism that MF can induce the expression changes of circRNAs in PDLSCs, which may modulate the OTM process and the alveolar bone remodeling.

In situ expression and serum level of thymic stromal lymphopoietin in oral lichen planus.

BACKGROUND: Oral lichen planus (OLP) is a chronic inflammatory disease of oral mucosa in which the CD8(+) T cell-mediated cytotoxicity is regarded as a major mechanism of pathogenesis. The main objective of this study is to investigate in situ expression and secretion of thymic stromal lymphopoietin (TSLP) in specimens and sera from patients with oral lichen planus. METHODS: Thirty-six patients with OLP and 35 donors enrolled in specimen and serum collection. Immunohistochemical method and immunofluorescence double-staining method were used to detect the expression of thymic stromal lymphopoietin and its receptor (TSLPR) together with CD8 in OLP specimens. Enzyme-linked immunosorbent assay (ELISA) was used to detect TSLP secretion. RESULTS: More TSLP- or TSLPR-positive cells showed in OLP specimens than in normal controls, and TSLP-positive cells were mainly in the epithelium, while TSLPR-positive cells mainly in the lamina propria. Furthermore, the number of TSLP-positive cells in the stratum basal was associated with the amount of mononuclear cells infiltrating in the lamina propria of OLP specimens. Among infiltrating mononuclear cells in the lamina propria, some CD8-positive cells also expressed TSLPR. The TSLP serum level of patients with OLP was significantly higher than of healthy donors, but there was no statistically difference between two clinical subtypes of OLP. CONCLUSIONS: Our findings provided the first evidence that TSLP may enroll in the pathology of OLP and the TSLP-TSLPR interaction may play an important role in it.

Effects of allogenous periosteal-derived cells transfected with adenovirus-mediated BMP-2 on repairing defects of the mandible in rabbits.

PURPOSE: This report describes the effect of periosteal-derived cells transfected with adenovirus-mediated bone morphogenetic protein-2 (BMP-2) on the repair of mandibular defects in rabbits. MATERIALS AND METHODS: Periosteal-derived cells were transfected with a replication-defective adenoviral vector encoding BMP-2, and the expression of BMP-2 was examined in transfected cells using in situ hybridization and enzyme-linked immunosorbent assay. In addition, the proliferation ability and activity of alkaline phosphatase of transfected cells were examined using the 3-[4,5-dimethylthiazol-2-Yl]-2,5-diphenyltetrazolium bromide method and enzymology, respectively. In vitro critical-size defects (about 10 × 6 mm) were made bilaterally in each rabbit mandible, and individual sites were implanted with tissue-engineered bone modified with an adenovirus construct encoding the recombinant human BMP-2 gene (Ad-BMP-2), tissue-engineered bone without modification, single bioactive glass ceramic, or no implants (control). New bone formation was evaluated by histochemical stain. RESULTS: BMP-2 expression in the supernate of infected cells was detected from the first day after Ad-BMP-2 transfection and remained at a high level for at least 2 weeks. Alkaline phosphatase expression in transfected cells was significantly greater than in uninfected cells. The group of Ad-BMP-2-modified periosteal-derived cells formed more new bone than the other group at any time point. CONCLUSION: Gene-modified tissue-engineered bone grafts have greater osteogenic potential than single tissue-engineered bone and single bioactive glass ceramic graft. Ex vivo Ad-BMP-2 transfer to periosteal-derived cells can increase bone formation in critical-size bone defects. Further studies are needed to determine if modified engineered cells can be developed for safe and effective clinical applications.

Dysbiotic tumor microbiota associates with head and neck squamous cell carcinoma outcomes.

BACKGROUND: The need for an effective tool to predict prognosis of head and neck squamous cell carcinoma (HNSCC) patients is critical and unmet. Microbiota has recently been found involved in tumor progression and response to immunotherapy. However, the association of microbiota with the prognosis of HNSCC patients remains obscure. This study aims to investigate the association between tumor microbiota and outcomes of HNSCC patients. METHODS: A retrospective study including 129 primary tumors of HNSCC was conducted. Using 16S rRNA sequencing, the profile and the composition of tumor microbiota were measured and their associations with overall survival (OS) and disease free survival (DFS) were examined. RESULTS: We observed a reduced richness and enriched abundances of genera Schlegelella and Methyloversatilis in tumor microbiota of HNSCC patients with poor prognosis. However, a richer tumor microbiota with greater abundances of genera Bacillus, and Lactobacillus and Sphingomonas was characterized in the patients with favorable prognosis.The ratio of these differentially abundant taxa, microbial dysbiosis index (MDI), was significantly associated with OS (hazard ratio [HR], 4.67, 95% confidence interval [CI], 2.51 to 8.69,P < 0.001) and DFS (HR, 2.89; 95% CI, 1.74 to 4.80, P < 0.001) independently of age, tumor size, lymph node metastasis, differentiation and p16 status. The risk score of multivariate Cox regression exhibited an excellent performance for estimating three-year OS (AUC of 0.826). We also found a richer tumor microbiota was correlated with moderate peritumoral inflammatory infiltration. CONCLUSION: These results indicate that tumor microbiota associates with outcomes of HNSCC patients.

CD68+ Macrophage Infiltration Associates With Poor Outcome of HPV Negative Oral Squamous Carcinoma Patients Receiving Radiation: Poly(I:C) Enhances Radiosensitivity of CAL-27 Cells but Promotes Macrophage Recruitment Through HMGB1.

Patients with human papillomavirus (HPV) negative oral squamous cell carcinoma (OSCC) generally have poor clinical outcomes and worse responses to radiotherapy. It is urgent to explore the underlining mechanisms of the distinct prognoses between HPV negative and HPV positive OSCC and to develop effective therapy strategy to increase the survival rate of HPV negative OSCC patients. We conducted a retrospective cohort of 99 resected OSCC patients to evaluate the prognosis of HPV negative and HPV positive OSCC patients receiving radiation or not. We further addressed the association of CD68+ macrophage infiltration with HPV status and the effects on survival of OSCC patients. We also used the TCGA-OSCC cohort for further verification. Based on the cohort study, we applied a synthetic dsRNA polymer, polyriboinosinic-polyribocytidylic acid (poly(I:C)), on CAL-27 (HPV negative OSCC cells). We co-cultured its condition medium with THP-1 derived macrophage and examined the cytokines and macrophage migration. We found that high CD68+ macrophage infiltration associated with poor overall survival in HPV negative OSCC patients receiving radiation. In vitro, poly(I:C) could induce apoptosis and enhance the radiosensitivity, but increase macrophage recruitment. Targeting HMGB1 could inhibit IL-6 induction and macrophage recruitment. Our findings indicated that CD68+ macrophage might play an important role in the outcomes of HPV negative OSCC patients receiving radiation. Our findings also suggested that radiation combined poly(I:C) might be a potential therapy strategy to increase the radiation response and prognosis of HPV negative OSCC. Notably, HMGB1 should be targeted to inhibit macrophage recruitment and enhance overall therapy effects.

Fucoidan reduced the invasion of oral squamous cell carcinoma cells and modified their effects to macrophages.

Fucoidan is a complex of polysaccharides showing antitumor and immunomodulation properties. Our previous studies found its regulation to myeloid immune cells, including macrophages. Aberrant infiltration and functions of macrophages are commonly found in oral squamous cell carcinoma (OSCC). In this study, we analyzed the effects of fucoidan on invasion of OSCC cells, and their regulation to macrophages, trying to evaluate its role as a potential therapy for OSCC. CAL27 and THP-1-derived macrophages were used as models for OSCC cells and tumor-infiltrated macrophages in the in vitro study, respectively. The effects of fucoidan on invasion of OSCC cells and their recruitment to macrophages were analyzed by transwell assay. KIF4A siRNA transfection was performed to investigate its role in fucoidan-modulated OSCC cells invasion. CCL3-neutralizing antibody was added into the conditioned medium of OSCC cells to evaluate its role in fucoidan-mediated macrophages recruitment and re-education. Fucoidan reduced the invasive potential of CAL27 cells with a decrease of MMP-2 and KIF4A transcription. KIF4A knockdown in CAL27 cells led to decreased invasion and MMP-2 expression. The conditioned medium of fucoidan-treated CAL27 cells promoted recruitment and inflammatory cytokines secretion on THP-1-derived macrophages. Further analysis found that fucoidan increased CCL3 production in CAL27 cells. Blocking CCL3 expression reversed the effects of fucoidan on macrophage recruitment and re-education. Our study found that fucoidan regulated the invasion of OSCC cells and also their recruiting and re-educating effects on macrophages, suggesting it could be a complementary approach in the treatment of OSCC.

Ultrasonic irradiation assisted surface modification of titanium plates to improve MC3T3-E1 cell proliferation.

The surface modification of titanium implants by NaOH has been widely investigated to improve their biocompatibility and bioactivity. It is very important to prepare an even network structure on titanium implants. In this work, ultrasonic irradiation (UI) is used along two different routes to obtain several modified surfaces on titanium plates: (1) the plates are first treated by a NaOH solution, and then UI is used to wash them in double distilled water; (2) the plates are modified by a NaOH solution in an ultrasonic cleaner with UI at 50 W. It is demonstrated that the UI energy can easily remove any weakly bound layers (WBL) on the titanium surface, leaving a strongly bound layer (SBL). The SBL is shown to enhance the proliferation of MC3T3-E1 preosteoblasts in comparison with the WBL. A dense and uniformly distributed nanostructure layer can be synthesized in situ on the surface of metallic titanium through a reaction between a NaOH solution and titanium under UI. The titanium plates prepared with NaOH and UI show better proliferation of preosteoblasts than titanium without UI.

[Preliminary study on transformation of the biological function of the cryopreserved osteoblasts cultured in vitro].

OBJECTIVE: To culture and amplify the young rabbit's bone marrow stromal cells (BMSCs) in vitro, and to observe the effect of hypothermia on the cells' growing behavior and biological function. METHODS: BMSCs were acquired from the rabbit' tibia bone marrow and induced to mature osteoblasts in vitro. The cultured cells growing well in vitro were preserved in liquid nitrogen. The anabiotic cells having cryopreserved for 1 week were chosen as the experimental group, and the routine 7th generation as the control group. Their biological function in comparion by the examination of morphological changes, cells' proliferation ability, colone forming ratio, synthesis ability of ALP and protein, mineralized nodes forming ability were observed. RESULTS: As contrast to the control groups, the anabiotic cells also grew and proliferated well in vitro except a little more slowly than before. They had the similar general shape in all the time segments, but a little differences in cells' ultrastructure. The experimental groups also had the typical characters of mature osteoblasts, and high abilities of the synthesis of ALP and proteins. The statistic data showed that these two groups had no significant difference (P > 0.05). CONCLUSION: The cryopreserved osteoblasts had the same biological functions and the similar growing behaviors as before. These results suggest that it is practical to use the cryopreserved osteoblasts for further study on bone tissue engineering.

[Surgical treatment of obstructive sleep apnea hypopnea syndrome after velopharyngeal ring ligation procedure].

PURPOSE: To evaluate the effect of removal of ligation thread combined adenoidectomy in the treatment of obstructive sleep apnea hypopnea syndrome (OSAHS) after velopharyngeal ring ligation procedure (VRLP). METHODS: The upper airway was examined with dynamic fiberoptic pharyngoscope in six patients with cleft palate who were diagnosed as OSAHS by polysomnography(PSG) after VRLP. All the patients underwent removal of ligation thread and adenoidectomy. Three months after operation, they were recalled back and were examined with polysomnography and dynamic fiberoptic pharyngoscope. The preoperative and postoperative PSG data were analysed with t test and chi-square test to determine the statistical differences. RESULTS: In patients with OSAHS after VRLP, a ring scar was observed on the upper airway at the soft palate level and a remarkable narrow nasopharyngeal meatus was found mainly because of adenoidal hypertrophy. After removal of ligation thread and adenoidectomy, no velopharyngeal incompetence was found, the upper airway morphology was smooth, respiration disorder during sleeping was completely disappeared and sleep architecture became normal. There were significant differences between preoperative and postoperative PSG data. CONCLUSION: Removal of ligation thread combined adenoidectomy was an effective treatment for OSAHS after VRLP.

[The experimental study of cryopreserved osteoblasts combined with BGC repairing mandibular defects in rabbits].

OBJECTIVE: The purpose of this study was to observe the biological characteristics of cultured Periosteal-derived Osteoblasts (POBs) preserved in liquid nitrogen in vitro, and to preliminarily study the osteogenetic capability of bioactive glass ceramic (BGC) combined with POBs. METHODS: The cryopreserved cells were cultured in DMEM medium and examined by morphological and histological assay. The POBs growing well in vitro were seeded into the porous BGC materials. A week later, the combined materials were implanted into the bone defects of rabbits' mandibular, the control groups were implanted into the single BGC (no cells). After 2, 4, 8, 12 weeks of operation, the specimens were respectively excised and examined by X-ray and histological chemistry. RESULTS: The cryopreserved POBs grew well in vitro and also had the tipical characteristics of mature osteoblasts. Cultured with BGC materials, the cells could attach, grow and proliferate well on the surface of most endoporous. After 4 weeks of operation, the transplanted osteoblasts began to form new osteoid or bone tissue in most pores of implanted BGC, and the bone defects were repaired better and earlier. CONCLUSION: It was practical to use the cryopreserved osteoblasts for further study on bone tissue engineering. It suggested that the "life active" bone would get more application and play a more important role in bone restoration and reconstruction.