[Effect of zoledronic acid on mandibular socket healing process of mice at an early stage].

PURPOSE: To investigate how bisphosphonates affect early process of socket healing in mice model. METHODS: Eighteen 8-9 weeks C57BL/6 male mice were randomly divided into control and experimental group. Bisphosphonate-related osteonecrosis of the jaw（BRONJ）model was conducted by intraperitoneal injection of zoledronate（ZOL） and extraction of lower left first molar in mice. Three, five, seven day after surgery, the mice were sacrificed and paraffin-embedded slides were made. H-E staining was used to evaluate the gross condition. The distribution and amounts of osteoclasts were measured by TRAP staining. Finally, immunochemical staining was used to detect RUNX2 and CTSK level. All experiments were duplicated thrice, ImageJ was used for transformation of pictures and SPSS 20.0 software package was used for statistical analysis. RESULTS: In comparison with the control group, early process of socket healing in the experimental group was generally delayed. RUNX2,CTSK,TRAP expression level was decreased. On the whole, early bone remodeling process in ZOL injection group was suppressed. CONCLUSIONS: Zoledronic acid inhibited the migration of fibroblasts into socket, and reduced the expression of Runx2, hindering new bone formation. It also can reduce the expression of TRAP 3，5，7 days after tooth extraction and CTSK expression three days after operation, thus inhibiting the bone resorption function of osteoclasts.

[Expression of hypoxia inducible factor-1alpha during mouse mandibular first molar eruption and its significance].

PURPOSE: To investigate the expression of hypoxia inducible factor-1alpha (HIF-1α) during the eruption of mouse lower first molar, and to explore the correlation between its expression and the activity of osteoclasts. METHODS: Mouse mandibles were dissected from mice at day 1.5 to 14.5 after birth. They were then serially sectioned and stained respectively using hemotoxylin, tartrate-resistant acid phosphatase (TRAP) staining, and immunohistochemical staining. SPSS 25.0 software package was used for data analysis. RESULTS: During the eruption of mouse lower first molar, the thickness of the crown alveolar bone gradually decreased, so did the number of osteoclasts and the expression of HIF-1α. CONCLUSIONS: During the eruption of mouse lower first molar, the less expression of HIF-1α, the fewer osteoclasts there were. HIF-1α may be involved in the eruption of mouse lower first molar by regulating the activity of osteoclasts.

[Mechanism of participation of osteocytes in the formation of osteoclasts under hypoxia].

OBJECTIVE: To investigate the mechanism of the participation of osteocytes in the formation of osteoclasts under hypoxia. METHODS: The hypoxia culture system of osteocyte-like cell line MLO-Y4 was established by deferoxamine mesylate (DFO) in vitro. The proliferation of MLO-Y4 cells was examined by CCK-8 cell proliferation/toxicity assay. RAW264.7 cells were induced to osteoclasts by the conditioned medium containing the cultured MLO-Y4. Tartrate-resistant acid phosphatase (TRAP) staining was performed on day 7. Quantitative real-time fluorescence polymerase chain reaction, immunofluorescence, and Western blot were used to detect the expression levels of hypoxia-inducible factor (HIF)-1α and receptor activator of nuclear factor-κB ligand (RANKL) in MLO-Y4 under hypoxia. The effects of siHIF-1α on the expression levels of HIF-1α and RANKL in MLO-Y4 under the same conditions were detected. RESULTS: DFO (100 µmol·L⁻¹) promoted the proliferation of MLO-Y4 at 24 h, which decreased with time (P<0.01). After the addition of soluble sRANKL, the formation of osteoclasts was significantly increased in the DFO group (P<0.001). The expression of RANKL mRNA in MLO-Y4 under 100 µmol·L⁻¹ DFO increased first and then decreased with the duration of hypoxia. This expression reached a peak at 24 h (P<0.01). Hypoxia up-regulated the expression of HIF-1α and RANKL protein (P<0.01). Under hypoxia, siHIF-1α downregulated the expression of HIF-1α and RANKL (P<0.01). siHIF-1α also decreased the number of osteoclasts (P<0.01). CONCLUSIONS: Under hypoxia, MLO-Y4 could facilitate the formation of RANKL through upre-gulating the expression of HIF-1α protein, thereby accelerate the differentiation of RAW264.7 cells into osteoclasts.

HIF-1α facilitates osteocyte-mediated osteoclastogenesis by activating JAK2/STAT3 pathway in vitro.

Osteocytes, entrapped within the mineralized bone matrix, has been found to have numerous functions such as acting as an orchestrator of bone remodeling through regulation of both osteoclast and osteoblast activity and also functioning as an endocrine cell. Due to a specialized morphology and surrounding structure, osteocytes are more tolerant to hypoxia during osteoporosis, fracture, osteoarthritis, and orthodontic-orthognathic combination therapy. Hypoxia-inducible factor-1α (HIF-1α) is one of the master regulators of hypoxia reactions, playing an important role in bone modeling, remodeling, and homeostasis. This study aimed to investigate the pivotal functional role of HIF-1α in osteocytes initiating of bone remodeling under hypoxia. In the present study, the osteoclasts formation induced by RAW264.7 was significantly promoted in conditioned media (CM) from osteocytic MLO-Y4 exposed to hypoxia in vitro. Therefore, hypoxic MLO-Y4 cells simulated by 100 µmol/L CoCl2 or 2% O2 stably expressed HIF-1α proteins and upregulated the expression of receptor activator of nuclear factor-κB ligand (RANKL) at both the messenger RNA (mRNA) and protein level. Furthermore, with the Knockdown of HIF-1α, the expression of RANKL mRNA and protein decreased after transient transfection. In addition, the phosphorylation of Janus kinase 2 (JAK2) and signal transducer and activator of transcription (STAT3) was also correlated with HIF-1α and RANKL levels under hypoxia. Then AG490, a JAK2 inhibitor, inhibited p-JAK2, p-STAT3 and RANKL expression. It was possible that AG490 disturbed the contact of HIF-1α and RANKL by JAK2/STAT3 pathway, influencing osteoclastogenesis. Our findings suggested that HIF-1α promoted the expression of RANKL by activating JAK2/STAT3 pathway in MLO-Y4 cells, and enhanced osteocyte-mediated osteoclastic differentiation in vitro.

Role of CD133+ cells in tongue squamous carcinomas: Characteristics of 'stemness' in vivo and in vitro.

The objective of the present study was to determine the 'stemness' characteristics of CD133+ cells (harvested from the squamous cell tongue carcinoma Tca-8113 cell line) in vitro and to observe the tumourigenicity of the CD133+ cells in the bodies of NOD/SCID mice. Single cells from the Tca-8113 cell line were observed for multiplication capacity in vitro. The suspending and pelletizing phenomena of Tca-8113 cells in vitro were also observed, and the expression of CD133 in squamous cell carcinoma of the tongue was measured. The CD133+ cells from the Tca-8113 cell line were purified, and their multiplication capacity and differentiation potency were observed. The NOD/SCID mouse model was established, and the tumourigenicity of the CD133+ cells was determined. The Tca-8113 cells were observed to emerge in the form of suspending tumour spheres in squamous cell carcinoma of the tongue. Monoplasts with sustainable multiplication capacity accounted for ~5.32% of the spheres, and 0.95% of the CD133+ cells were expressed in squamous cell carcinoma of the tongue, with stronger multiplication capacity and differentiation potency in vitro. Stronger tumourigenicity was also observed in the bodies of the NOD/SCID mice. CD133- cells exhibited a multiplication capacity to a certain extent. Overall, the CD133+ cells in squamous cell carcinoma of the tongue are characterised by relatively strong tumourigenicity capacity in vivo and in vitro. To a certain extent, these CD133+ cells demonstrate the characteristics of 'stemness'.

Hypoxia-inducible factor-1α overexpression indicates poor clinical outcomes in tongue squamous cell carcinoma.

The aim of the present study was to investigate the expression of hypoxia-inducible factor-1α (HIF-1α) in tongue squamous cell carcinoma (TSCC) and to assess its possible impact on prognosis. A total of 49 tumor samples and 15 adjacent non-tumor samples from 49 patients treated between January 2000 and December 2005 at the Department of Oral and Maxillofacial Surgery, Hospital of Stomatology, Tongji University (Shanghai, China) were obtained for investigation with immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The expression of HIF-1α was detected in 87.76% (43/49) of the TSCC samples and in 33.33% (5/15) of the adjacent non-tumor tissues. The expression of vascular endothelial growth factor (VEGF) was also observed in 83.67% (41/49) of the TSCC samples and in only 20% (3/15) of the adjacent non-tumor samples at a low level. RT-PCR revealed that the mRNA expression of HIF-1α and VEGF was present in the tumor tissues; however, it was barely detected in the corresponding adjacent normal tissues. The overexpression of HIF-1α was significantly associated with T classification (P=0.01), lymphatic metastasis (P=0.05) and histological differentiation (P<0.001). Furthermore, HIF-1α overexpression was significantly associated with poor overall (P=0.001) and disease-free survival rates (P=0.01), independent of T stage and lymphatic metastasis. The Cox proportional hazards regression model demonstrated that the level of HIF-1α expression may be an independent prognostic factor for TSCC. HIF-1α overexpression was observed in TSCC and its overexpression suggests a poor prognosis. HIF-1α may be a molecular marker for predicting the prognosis of TSCC.

Effects of trichostatin A on HIF-1α and VEGF expression in human tongue squamous cell carcinoma cells in vitro.

Hypoxia is an essential feature of the microenvironment of solid tumors, which regulates a variety of transcription factors including hypoxia-inducible factor-1α (HIF-1α). HIF-1α overexpression enhances tumor angiogenesis via upregulation of vascular endothelial growth factor (VEGF) and some other hypoxia-inducible angiogenic factors, which lead to a more aggressive tumor phenotype, tumor metastasis and resistance to radiation and chemotherapy. In this study, we found that a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), inhibited cell proliferation and invasion, blocked the cell cycle, and induced cell apoptosis in a dose- and time-dependent manner in the human tongue squamous cell carcinoma (TSCC) SCC-6 cell line in vitro. Furthermore, TSA reduced both basal levels and hypoxia-induced HIF-1α protein accumulation but not HIF-1α mRNA levels, and both protein and mRNA levels of VEGF expression. These results showed that TSA had a potent anticancer activity on TSCC cells, suggesting that TSA could be a promising drug targeting tumor angiogenesis via inhibition of HIF-1α and VEGF expression in the development of an effective chemopreventive and anticancer agent on human TSCCs.

[Biological characteristics of CD133+ subpopulation in tongue squamous cell carcinoma Tca8113 cell line].

OBJECTIVE: To detect the expression of CD133 in human tongue squamous cell carcinoma Tea8113 cell line and observe proliferation ability of CD133 groups in vitro. METHODS: Limiting dilution assay was employed to observe the proliferating character of Tca8113 single cell in vitro. The ability of growing as cancer spheroids was observed with ultra-low attachment plates. The flow cytometry was used to detect the expression of putative tumor-initiating cell marker CD133 in human tongue squamous cell carcinoma Tca8113 cell line. The selective technique of immunomagnetic beads was applied to purify CD133 tumor cells, CD133 tumor cells were cultured and their ability of proliferation were observed in vitro. RESULTS: After 12 days, the result of single cell culture in vitro revealed that about 5.23% of cultured Tca8113 cells possessed the capacity of continue proliferation. The cells line fromed floating clusters with one week of passaging cells into non-adherent plates. Approximately 0.95% of cells in Tca8113 cell line expressed CD133. Compared with CD133- cells and control Tca8113 cells, CD133+ cells demonstrated increased proliferation capacity. The proportion of CD133 cells decreased in culture as days passed. The percentage of CD133+ cells decreased from 92.45% to 1.62% in twelve days' culture. CONCLUSION: Tumor stem cells have the character of heterogenity and lower proportion of CD133 but higher ability of proliferation, and the diferentiation in human tongue squamous cell carcinoma Tca8113 cell line in vitro, CD133 may be one of makers for tumor-initiating cell of human tongue squamous cell carcinoma Tca8113 cell line.

[Proteomics study of the serum biomarkers in tongue cancer].

OBJECTIVE: To search for specific serum biomarkers associated with tongue cancer by means of the serum proteomics technology. METHODS: The tongue cancer cells of human tongue cancer cell line Tca8113 were subcutaneously inoculated into nude mice, while control nude mice were injected with phosphate-buffered saline. Serums from these two group of mice were collected for proteomic analysis using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). RESULTS: Comparing the serum 2-DE maps from the tumor-bearing mice with those produced from control mice, we found that squamous cell carcinoma antigen 1 was over expressed only in the serum of tumor-bearing mice. CONCLUSIONS: The squamous cell carcinoma antigen 1 may be of great potential as the biomarker of tongue cancer and as the potential therapeutic target for gene therapy.

[Experimental studies on ectopic osteogenesis by seeding human mesenchymal stem cell on human natural bone derived scaffold materials].

OBJECTIVE: To investigate the ectopic osteogenesis potential of human natural bone derived material combined with human bone marrow mesenchymal stem cells (MSCs). METHODS: Cell-scaffold complexes were implanted subcutaneously into the left back of the nude mice, and human natural bone derived material were implanted into the right back as control group. The mice were killed respectively on the postoperative 2 weeks, 4 weeks and 8 weeks. The macroscopic, histopathological, alkaline phosphatase (ALP) activity assay methods were performed to assess the ectopic osteogenesis potential. RESULTS: The cartilaginous osteogenesis were observed in both deproteinated bone and decalcified bone, and the more new bone tissue formed gradually as the time went by after implantation. ALP activity become stronger followed with the time (P < 0.05), and compared with the decalcified bone, deproteinated bone displayed stronger ALP activity (P < 0.05). CONCLUSION: The MSCs and human natural bone derived material can be used as good seed cells and scaffold materials respectively to construct tissue-engineered bone, and as the scaffold material, deproteinated bone has better osteogenesis ability than decalcifed bone.