Assessment of the correlation between supracrestal gingival tissue dimensions and other periodontal phenotypes components via the digital registration method: a cross­sectional study in a Chinese population.

BACKGROUND: Supracrestal gingival tissue dimensions (SGTDs) has been considered to be an essential element of periodontal phenotype (PP) components. This study aimed to explore the relationship between SGTDs and other PP components by digital superposition method that integrated cone beam computed tomography (CBCT) with intraoral scanning. METHODS: This cross-sectional study was conducted at the Stomatology Hospital of Fujian Medical University. Participants were recruited based on the inclusion and exclusion criteria. The data obtained from the digital scanner (TRIOS 3, 3Shape, Denmark) and CBCT images were imported into the TRIOS software (Implant Studio, 3Shape, Denmark) for computing relevant parameters. The significant level was set at 0.05. RESULTS: A total of 83 participants with 498 maxillary anterior teeth were finally included. The mean values of supracrestal gingival height (SGH) and the distance from the cementoenamel junction (CEJ) to the crest of the alveolar ridge (CEJ-ABC) on the buccal site were significantly higher than palatal SGH (SGH-p) and palatal CEJ-ABC (CEJ-ABC-p). Men exhibited taller CEJ-ABC and SGH-p than women. Additionally, tooth type was significantly associated with the SGH, SGH-p and CEJ-ABC-p. Taller SGH was associated with wider crown, smaller papilla height (PH), flatter gingival margin, thicker bone thickness (BT) and gingival thickness (GT) at CEJ, the alveolar bone crest (ABC), and 2 mm apical to the ABC. Smaller SGH-p displayed thicker BT and GT at CEJ, the ABC, and 2 and 4 mm apical to the ABC. Higher CEJ-ABC showed lower interproximal bone height, smaller PH, flatter gingival margin, thinner GT and BT at CEJ, and 2 mm apical to the ABC. Smaller CEJ-ABC-p displayed thicker BT at CEJ and 2 and 4 mm apical to the ABC. On the buccal, thicker GT was correlated with thicker BT at 2 and 4 mm below the ABC. CONCLUSION: SGTDs exhibited a correlation with other PP components, especially crown shape, gingival margin and interdental PH. The relationship between SGTDs and gingival and bone phenotypes depended on the apico-coronal level evaluated. TRIAL REGISTRATION: This study was approved by the Biomedical Research Ethics Committee of Stomatology Hospital of Fujian Medical University (approval no. 2023-24).

Effects and mechanisms of Porphyromonas gingivalis outer membrane vesicles induced cardiovascular injury.

BACKGROUND: The outer membrane vesicles (OMVs) derived from Porphyromonas gingivalis (P. gingivalis) have long been acknowledged for their crucial role in the initiation of periodontitis. However, the implications of P. gingivalis OMVs in the context of cardiovascular disease (CVD) remain incompletely understood. This study aimed to clarify both the impact and the underlying mechanisms through which P. gingivalis OMVs contribute to the propagation of distal cardiovascular inflammation and trauma. METHODS: In this study, various concentrations (0, 1.25, 2.5, and 4.5 µg/µL) of P. gingivalis OMVs were microinjected into the common cardinal vein of zebrafish larvae at 48 h post-fertilization (hpf) to assess changes in cardiovascular injury and inflammatory response. Zebrafish larvae from both the PBS and the 2.5 µg/µL injection cohorts were harvested at 30 h post-injection (hpi) for transcriptional analysis. Real-time quantitative PCR (RT-qPCR) was employed to evaluate relative gene expression. RESULTS: These findings demonstrated that P. gingivalis OMVs induced pericardial enlargement in zebrafish larvae, caused vascular damage, increased neutrophil counts, and activated inflammatory pathways. Transcriptomic analysis further revealed the involvement of the immune response and the extracellular matrix (ECM)-receptor interaction signaling pathway in this process. CONCLUSION: This study illuminated potential mechanisms through which P. gingivalis OMVs contribute to CVD. It accentuated their involvement in distal cardiovascular inflammation and emphasizes the need for further research to comprehensively grasp the connection between periodontitis and CVD.

Demineralized dentin matrix promotes gingival healing in alveolar ridge preservation of premolars extracted for orthodontic reason: a split-mouth study.

Objective: The purpose of this study was to prospectively evaluate the efficacy of a demineralized dentin matrix (DDM) in decreasing the initial inflammatory response of the gingiva and facilitating the repair and regeneration of soft tissue in alveolar ridge preservation. Methods: This clinical study employed a split-mouth design. Fourteen patients with a total of forty-four sites underwent extraction and alveolar ridge preservation (ARP) procedures. A Bilaterally symmetrical extraction operation were conducted on the premolars of each patient. The experimental group received DDM as a graft material for ARP, while the control group underwent natural healing. Within the first month postoperatively, the pain condition, color, and swelling status of the extraction sites were initially assessed at different time points Subsequently, measurements were taken for buccal gingival margin height, buccal-lingual width, extraction socket contour, and the extraction socket area and healing rate were digitally measured. Additionally, Alcian Blue staining was used for histological evaluation of the content during alveolar socket healing. Results: Both groups experienced uneventful healing, with no adverse reactions observed at any of the extraction sites. The differences in VAS pain scores between the two groups postoperatively were not statistically significant. In the early stage of gingival tissue healing (3 days postoperatively), there were statistically significant differences in gingival condition and buccal gingival margin height between the two groups. In the later stage of gingival tissue healing (7, 14, and 30 days postoperatively), there were statistically significant differences in buccal-lingual width, extraction socket healing area, and healing rate between the two groups. Furthermore, the histological results from Alcian Blue staining suggested that the experimental group may play a significant role in promoting gingival tissue healing, possibly by regulating inflammatory responses when compared to the control group. Conclusion: The application of DDM in alveolar ridge preservation has been found to diminish initial gingival inflammation after tooth extraction. Additionally, it has shown the ability to accelerate early gingival soft tissue healing and preserve its anatomical contour. Clinical trial registration: chictr.org.cn, identifier ChiCTR2100050650.

Corrigendum: Demineralized dentin matrix promotes gingival healing in alveolar ridge preservation of premolars extracted for orthodontic reason: a split-mouth study.

[This corrects the article DOI: 10.3389/fendo.2023.1281649.].

The regulation mechanism of LINC00707 on the osteogenic differentiation of human periodontal ligament stem cells.

The osteogenic differentiation of periodontal ligament stem cells (PDLSCs) is important for periodontal tissue repair and regeneration. Long non-coding RNAs (lncRNAs) are key regulators of diverse biological processes. However, their roles in PDLSC osteogenic differentiation are still largely unknown. This study explored the effect of LINC00707 and its mechanism on the osteogenic differentiation of human PDLSCs. Results showed an increase in LINC00707 and forkhead box O1 (FOXO1) but a decrease in miR-490-3p during PDLSC osteogenic differentiation. LINC00707 and FOXO1 promoted osteogenic differentiation as evidenced by the formation of calcium nodules and the increase in osteogenic markers such as alkaline phosphatase, osteocalcin (OCN), and runt-related transcription factor 2 (Runx2). LINC00707 and FOXO1 knockdown exhibited opposite effects. Dual-luciferase reporter assay and qRT-PCR showed that LINC00707 can specially bind to miR-490-3p, which reversed the effect of LINC00707 on PDLSCs. MiR-490-3p inhibitor relieved the inhibiting effect of sh-LINC00707 on osteogenic differentiation. Further investigation revealed that LINC00707 can promote osteogenic differentiation by regulating FOXO1 expression through miR-490-3p sponging. Thus, the LINC00707/miR-490-3p/FOXO1 axis modulated PDLSC osteogenic differentiation and might be a promising therapeutic target for periodontal diseases.

LncRNA MEG3 Inhibits the Degradation of the Extracellular Matrix of Chondrocytes in Osteoarthritis via Targeting miR-93/TGFBR2 Axis.

BACKGROUND: As a degenerative joint disease, osteoarthritis (OA) is characterized by articular cartilage degradation. Long noncoding RNAs (lncRNAs) act critical roles in the regulation of OA development, including affecting the proliferation, apoptosis, extracellular matrix (ECM) degradation, and inflammatory response of chondrocytes. The current study's aim was to investigate the regulatory function and the underlying molecular mechanism of lncRNA MEG3 in ECM degradation of chondrocytes in OA. METHODS: In the current study, chondrocytes were induced by interleukin-1ß (IL-1ß) to simulate OA condition, and further assessed cell viability, lncRNA MEG3 and miR-93 expression levels. Overexpression or knockdown of lncRNA MEG3 in chondrocytes treated with IL-1ß were performed to investigate the function of MEG3 in regulating cell proliferation, apoptosis and ECM degradation using EdU assay, flow cytometry, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and Western blot. The interaction between MEG3 and miR-93 was assessed using qRT-PCR. Furthermore, overexpression of miR-93 was performed as recovery experiment to explore the functional mechanism of MEG3. RESULTS: MEG3 was significantly downregulated in chondrocytes treated with IL-1ß, whereas miR-93 was upregulated concomitantly. Overexpression of MEG3 induced the proliferation, suppressed the apoptosis, and relieved the degradation of ECM in IL-1ß-induced chondrocytes. By contrast, knockdown of MEG3 suppressed the proliferation, promoted the apoptosis, and aggravated ECM degradation in IL-1ß induced chondrocytes. In addition, MEG3 was found to relieve the inhibitive expression of TGFBR2 as a competitive endogenous RNA (ceRNA) of miR-93, and then activated transforming growth factor-ß (TGF-ß) signaling pathway, regulated chondrocytes ECM degradation in IL-1ß induced chondrocytes subsequently. CONCLUSION: LncRNA MEG3 targeted miR-93/TGFBR2 axis, regulated the proliferation, apoptosis and ECM degradation of chondrocytes in OA.

MicroRNA-675-3p regulates IL-1ß-stimulated human chondrocyte apoptosis and cartilage degradation by targeting GNG5.

Growing evidence has indicated that microRNAs (miRNAs) are modulators of osteoarthritis (OA) development and progression. In this study, we first evaluated the anti-apoptosis and chondroprotective effects of microRNA-675-3p (miR-675-3p) on interleukin-1ß (IL-1ß)-stimulated human chondrocytes. The overexpression of miR-675-3p inhibited apoptosis and cartilage matrix degradation and promoted cell proliferation in human chondrocytes. Target gene prediction and luciferase reporter assays suggested that G-protein subunit γ 5 (GNG5) may be the target gene of miR-675-3p. The overexpression of miR-675-3p inhibited IL-1ß-stimulated chondrocyte apoptosis, and this effect was reversed by the overexpression of GNG5. Finally, we used bioinformatic tools and biological methods to show that the long noncoding RNA X-inactive specific transcript (lncRNA XIST) could bind to miR-675-3p, which affects the expression of GNG5 mRNA. Our findings may substantiate miR-675-3p as a new treatment for OA.

Nicotinamide-induced silencing of SIRT1 by miR-22-3p increases periodontal ligament stem cell proliferation and differentiation.

Dental stem cell proliferation and osteoblast differentiation are key cellular processes involved in periodontitis diseases. Researchers have found that SIRT1 (sirtuin 1, silent mating type information regulation 2 homolog 1) and microRNAs play a pivotal role in the process, but a clear underlying mechanism has not been determined. In this study, the has-miR-22-3p that target SIRT1 was predicted by TargetScan. Luciferase reporter assay was used to confirm that SIRT1 is the direct target of miR-22-3p. Importantly, miR-22-3p was revealed to control SIRT1 in periodontal ligament stem cell (PDLSC) and to regulate the proliferation and differentiation of PDLSC by SIRT1 silencing. Furthermore, we detected the induction of miR-22-3p expression by nicotinamide treatment on PDLSC. Induction of PDLSC proliferation and differentiation by nicotinamide treatment was blocked by miR-22-3p knockdown. These results suggested that the effect of nicotinamide on PDLSC is through miR-22-3p. In addition, miR-22-3p also upregulated the expression levels of the inflammatory cytokines tumor necrosis factor-α, interleukin-1ß (IL-1ß), and IL-8 in PDLSC through SIRT1 pathway and downregulated the expression of TLR-2 and TLR-4. miR-22-3p is a new target either for the treatment of periodontitis or the improvement of inflammation caused by orthodontics.

Knockdown Of lncRNA NCK-AS1 Regulates Cisplatin Resistance Through Modulating miR-137 In Osteosarcoma Cells.

PURPOSE: Long non-coding RNAs (lncRNAs) have been proved to act crucial parts in the progress of human tumor. However, the role of lncRNAs in drug resistance of tumor cells remains to be further elucidated. The present study aimed to explore whether lncRNA NCK-AS1 could affect the cisplatin (DDP) resistance in human osteosarcoma cell and the underlying molecular mechanism. METHODS: The expression of NCK1-AS1 and miR-137 in osteosarcoma cells was detected by qRT-PCR. CCK-8 assay, colony formation assay, Western blotting, wound healing assay and transwell assay were employed to assess the cell proliferation, migration and invasion. In addition, CCK-8 assay, flow cytometry, qRT-PCR and resistance gene activity analysis were performed to assess the DDP sensitivity of osteosarcoma cells. The interaction between NCK1-AS1 and miR-137 was identified using a dual-luciferase reporter gene assay and RNA immunoprecipitation (RIP) assay. RESULTS: The results revealed that NCK1-AS1 was significantly upregulated in osteosarcoma cells, as well as in DDP-resistant osteosarcoma cells. NCK1-AS1 silence inhibited the proliferation, migration and invasion of osteosarcoma cells, whereas enhanced the sensitivity of osteosarcoma cells to DDP. Furthermore, NCK1-AS1 directly interacted with miR-137 and overexpression of miR-137 suppressed the proliferation, migration and invasion of osteosarcoma cells. Most importantly, miR-137 overexpression enhanced the sensitivity of osteosarcoma cells to DDP, and high expression of NCK1-AS1 reversed the influences of miR-137 overexpression on DDP-resistant cells. CONCLUSION: In short, NCK1-AS1 knockdown enhanced DDP sensitivity of osteosarcoma cells by regulating miR-137, which may be a novel potential target for anti-DDP resistance in human osteosarcoma.

The effects of TRAF6 on proliferation, apoptosis and invasion in osteosarcoma are regulated by miR-124.

The present study aimed to verify tumor necrosis factor receptor­associated factor 6 (TRAF6) as the target gene of microRNA-124 (miR-124). In addition, the expression of miR­124 was investigated in osteosarcoma tissues and cells, and its effects on the biological characteristics of osteosarcoma cells were determined, in order to provide an experimental and theoretical basis for the application of TRAF6 in the treatment of osteosarcoma. A fluorescence reporter enzyme system was used to verify TRAF6 as a target gene of miR­124, and western blotting was used to detect the effects of miR­124 on the protein expression levels of TRAF6 in cells. The expression levels of miR­124 were detected in osteosarcoma tissues and an osteosarcoma cell line (MG­63) by quantitative polymerase chain reaction (qPCR). In addition, a total of 48 h post­transfection of MG­63 cells with a miR­124 mimic, qPCR was used to detect the expression levels of miR­124, and the effects of miR­124 on the viability of MG­63 human osteosarcoma cells was determined using the MTT method. The effects of miR­124 on the cell cycle progression and apoptosis of MG­63 cells were analyzed by flow cytometry, whereas the effects of miR­124 on the migration of MG­63 cells was detected using the Transwell invasion chamber analysis method. A TRAF6 recombinant expression plasmid (pcDNA3.1­TRAF6) was also constructed, and MG­63 cells were transfected with the recombinant plasmid and a miR­124 mimic, in order to further validate the biological role of miR­124 via the regulation of TRAF6. The results of the present study indicated that, compared with in the normal control group, the expression levels of miR­124 were significantly increased in MG­63 cells transfected with a miR­124 mimic (P<0.01). In addition, the luciferase reporter gene system demonstrated that, compared with in the control group, relative luciferase activity was significantly reduced in the miR­124 mimic group (P<0.01). The results of MTT analysis indicated that cell viability was also significantly reduced in response to the overexpression of miR­124 in MG­63 cells (P<0.01). Flow cytometric analysis demonstrated that the proportion of cells in S phase and G2/M phase was significantly decreased (P<0.01) in cells overexpressing miR­124, and the number of apoptotic cells was significantly increased (P<0.01). Furthermore, the results of the Transwell invasion assay suggested that the number of invasive cells was significantly decreased following enhanced expression of miR­124 (P<0.01). In MG­63 cells overexpressing miR­124 and TRAF6, the results of MTT, flow cytometric and Transwell assay analyses demonstrated that the overexpression of TRAF6 had the opposite biological effects compared to miR­124 overexpression. In conclusion, the present study indicated that the expression levels of miR­124 were downregulated in human osteosarcoma tissues and cells, and that miR­124 is associated with negative regulation of TRAF6 expression; therefore, the role of TRAF6 in primary osteosarcoma may be regulated by miR­124. Therapeutic strategies that enhance miR­124 expression or inhibit TRAF6 expression may be beneficial for the treatment of patients with osteosarcoma.

MicroRNA-19 contributes to the malignant phenotypes of osteosarcoma in vitro by targeting Pax6.

This study was conducted to detect the expression of miR-19 and Pax6 (Paired box protein 6) in human osteosarcoma cells and the effects on biological characteristics of osteosarcoma cells. Quantitative real-time polymerase chain reaction was used to detect the expression of Pax6 and miR-19 in normal human osteoblasts (hFOB 1.19) and osteosarcoma cell lines (U2OS, Saos-2, and MG-63). Results showed that miR-19 was significantly upregulated in osteosarcoma cell lines compared with that in hFOB 1.19 cells, while the expression of Pax6 messenger RNA was significantly downregulated. Pax6 was defined as the target gene of miR-19 which was validated by luciferase reporter gene analysis. Results indicated that miR-19 had an interaction with Pax6 3'-untranslated region. At the same time, the protein expression of Pax6 was significantly decreased in the MG-63 cells transfected with miR-19 mimic and was notably enhanced in osteosarcoma MG-63 cells transfected with miR-19 inhibitor. These data suggested that Pax6 was a target of miR-19 in osteosarcoma MG-63 cells. The effects of miR-19 on the biological behavior of MG-63 cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry, and Transwell assay. Results showed that the downregulation of miR-19 inhibited cell viability, reduced the percentage of cells in S phase and the number of cells passing through the Transwell chamber, and increased the number of apoptotic cells. Western blot analysis showed that the inhibition of miR-19 significantly increased the expression of epithelial proteins (E-cadherin and ß-catenin) and decreased the expression of mesenchymal protein (Vimentin), extracellular signal-regulated kinase, and phosphorylated extracellular signal-regulated kinase in MG-63 cells. MiR-19 inhibitor and Pax6 small interfering RNA were simultaneously transfected into MG-63 cells. Results from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry, and Transwell assay demonstrated that the inhibition of Pax6 expression in MG-63 cells could reverse the cell biological effects induced by the inhibition of miR-19 expression. Based on these findings, it was suggested that miR-19, upregulated in osteosarcoma cells, negatively regulated the expression of Pax6, which can promote the malignant phenotypes of osteosarcoma cells via activation of the extracellular signal-regulated kinase signaling pathways. Therefore, miR-19/Pax6 may offer potential for use as a target for the treatment of osteosarcoma.

Spatiotemporal expression of FOXA1 correlates with reactive gliosis after spinal cord injury.

Forkhead box A1 (FOXA1) is a member of the FOX family of transcription factors and involved in various mammalian processes. However, the expression and function of FOXA1 in central nervous system (CNS) are still with limited acquaintance. In present study, we performed an acute spinal cord injury (SCI) model in adult rats and investigated the dynamic changes of FOXA1 expression in spinal cord. We found that FOXA1 protein levels were significantly increased after SCI and we observed that the expression of FOXA1 is enhanced in the white matter. Meanwhile, double immunofluorescence staining showed that increased levels of FOXA1 were striking in astrocytes and microglia. We also examined the expression of proliferating cell nuclear antigen (PCNA), whose changes were correlated with the expression profiles of FOXA1. In vitro, FOXA1 depletion by siRNA inhibited astrocyte proliferation and migration. Meanwhile, FOXA1 knockdown also reduce cell cycle related proteins. Which indicated that FOXA1 might modulate cell cycle progression and play a crucial role in cell proliferation. Furthermore, FOXA1 knockdown also inhibited LPS-induced synthesis/secretion of IL-1ß and TNF-α in primary microglia. These results indicated that FOXA1 might play an important role in pathophysiology after SCI.

Antitumor activity of dobutamine on human osteosarcoma cells.

Dobutamine has been widely used for the treatment of heart failure and cardiogenic shock since the 1970s. Osteosarcoma is the most commonly observed malignant bone tumor in children. Currently, there are no effective drugs for the treatment of osteosarcoma. In the present study, the potential anticancer activity of dobutamine on human osteosarcoma cells was examined. Human osteosarcoma MG-63 cells were treated with dobutamine at various concentrations and for various incubation times. The inhibition of cell growth by dobutamine was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Flow cytometry was utilized to evaluate the effect of dobutamine on cell apoptosis and the cell cycle. Furthermore, the expression levels of caspase-3 and caspase-9 were assessed by western blot analysis. The influence of dobutamine on cancer cell migration and invasion was additionally evaluated using wound-healing assay and the Boyden Chamber migration method. Dobutamine significantly inhibited the growth of MG-63 cells at a concentration of 10 µM or higher when incubated for 12 h or longer (P=0.023). Dobutamine augmented cell apoptosis and arrested the cell cycle in the G2/M phase. Western blot analysis revealed that dobutamine induces expression of caspase-3 and caspase-9. In addition, the invasiveness and migration of MG-63 cells was inhibited by dobutamine in a concentration-dependent manner. The results of the present study may lead to novel applications for dobutamine in the treatment of osteosarcoma.

HMGB1 promotes cellular proliferation and invasion, suppresses cellular apoptosis in osteosarcoma.

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Unfortunately, treatment failures are common due to the metastasis and chemoresistance, but the underlying molecular mechanism remains unclear. Accumulating evidence indicated that the deregulation of DNA-binding protein high-mobility group box 1 (HMGB1) was associated with the development of cancer. This study aimed to explore the expression of HMGB1 in osteosarcoma tissues and its correlation to the clinical pathology of osteosarcoma and to discuss the role of HMGB1 in the development of osteosarcoma. The results from RT-PCR and Western blot showed that the expression rate of HMGB1 messenger RNA (mRNA) and the expression of HMGB1 in the osteosarcoma tissues were significantly higher than those in normal bone tissue (p < 0.05), the expression rate of HMGB1 mRNA and the expression of HMGB1 in the carcinoma tissues with positive lung metastasis were significantly higher than those without lung metastasis (p < 0.05), and with increasing Enneking stage, the expression rate of HMGB1 mRNA and the expression of HMGB1 also increased (p < 0.05). In order to explore the role of HMGB1 in osteosarcoma, the expression of HMGB1 in the human osteosarcoma MG-63 cell line was downregulated by the technique of RNA interference. Western blot results showed that the protein expression of HMGB1 was significantly decreased in the MG-63 cells from HMGB1-siRNA transfection group (p < 0.05), which suggested that HMGB1 was successfully downregulated in the MG-63 cells. Then the changes in proliferation, apoptosis, and invasion of MG-63 cells were examined by MTT test, PI staining, annexin V staining, and transwell chamber assay. Results showed that the abilities of proliferation and invasion were suppressed in HMGB1 knockdown MG-63 cells, and the abilities of apoptosis were enhanced in HMGB1 knockdown MG-63 cells. The expression of cyclin D1, MMP-9 was downregulated in HMGB1 knockdown MG-63 cells, and the expression of caspase-3 was upregulated in HMGB1 knockdown MG-63 cells. Taken together, the overexpression of HMGB1 in osteosarcoma might be related to the tumorigenesis, invasion, and metastasis of osteosarcoma, which might be a potential target for the treatment of osteosarcoma.

Spatiotemporal pattern of TRAF3 expression after rat spinal cord injury.

TNF receptor associated factor 3 (TRAF3), a member of the TRAF family of intracellular signaling proteins, can directly influence the phosphorylation status and activation of c-Jun N-terminal kinase, participating in CD40-induced apoptosis in carcinoma. However, its expression profile and function are still unclear in spinal cord injury (SCI). In this study, we performed an acute spinal cord contusion injury model in adult rats and detected the dynamic change patterns of TRAF3 expression in spinal cord. Western blot and immunohistochemistry revealed a striking upregulation of TRAF3 after SCI. Double immunofluorescence staining prompted that TRAF3 immunoreactivity was found in neurons rather than astrocytes. Moreover, co-localization of TRAF3/active caspase-3 was detected in neuronal nuclei. To further investigate the function of TRAF3, a neuronal cell line PC12 was employed to establish an apoptosis model in vitro. We analyzed the association of TRAF3 with active caspase-3 on PC12 cells by western blot and immunofluorescent labeling, which was parallel with the data in vivo. Additionally, knocking TRAF3 down with siRNA demonstrated the probable pro-apoptotic role of TRAF3 in the process of neuronal apoptosis. To summarize, we firstly uncover the temporal and spatial expression changes of TRAF3 in SCI. Our data suggest that TRAF3 might be implicated in central nervous system pathophysiology after SCI.

Operative strategy for different types of thoracolumbar stress fractures in ankylosing spondylitis.

STUDY DESIGN: There are no accurate guidelines on the operative treatment of ankylosing spondylitis (AS)-related thoracolumbar stress fractures. For this reason, we categorized such bone fractures into 2 types: vertebral body type and intervertebral space type, according to the damage mechanism, cross-section spot, and iconography, and devised a targeted surgical plan based on the characteristics of each fracture type. OBJECTIVE: To investigate the types and surgical treatment of thoracolumbar stress fractures in AS. SUMMARY OF BACKGROUND DATA: Thoracolumbar stress fractures are complications of AS. The patients with AS have a higher fracture risk that is approximately 3.5 times than the healthy. As the mechanism of injury, clinical manifestations, imaging characteristics, and principles of treatment of these fractures differ from those of general spinal fractures, the surgical approach is different from that of AS kyphosis orthopedic surgery. In this study, we summarize the clinical data of 11 AS patients with thoracolumbar fractures and discuss the clinical efficacy of the surgical approach based on the mechanism of injury and radiographic features. METHODS: We reviewed the data of 11 patients who underwent surgery for AS-related thoracolumbar stress fractures. Five patients with vertebral body-type fractures underwent vertebral wedge osteotomy through the pedicle and posterior internal fixation, whereas 6 patients with intervertebral space-type fractures underwent anterior spinal decompression with strut grafting and posterior internal fixation. RESULTS: The follow-up period was 2-4 years. After surgery, all kyphosis deformities were corrected, low back pain was relieved immediately, and scores on the visual analog scale improved by >70%. At the final follow-up, kyphosis correction had no significant loss. In the radiographic images, neither nonunion signs of pseudarthrosis plane nor neurologic or infectious complications were observed. CONCLUSIONS: Choosing an anterior or posterior surgical approach based on the type of AS-related thoracolumbar stress fracture can enhance the stability of the fracture, effectively restore the neurocanal volume and spinal column's axis of stress, reduce the complications of spinal cord damage, and produce the desired clinical curative effect.

SASH1 regulates proliferation, apoptosis, and invasion of osteosarcoma cell.

SASH1, a member of the SLY-family of signal adapter proteins, is a candidate tumor suppressor in breast and colon cancer. The SASH1 protein possesses both the SH3 and SAM domains, indicating that it may play an important role in intracellular signal transduction. Reduced expression of SASH1 is closely related to tumor growth, invasion, metastasis, and poor prognosis. However, the biological role of SASH1 remains unknown in osteosarcoma. To unravel the function of SASH1, we explored the expression of SASH1 in osteosarcoma tissues and its correlation to the clinical pathology of osteosarcoma and analyzed the relationship between SASH1 expression and cell cycle, apoptosis and invasion of osteosarcoma MG-63 cells, using the flow cytometry analysis and transwell invasion chamber experiments. Furthermore, the effect of SASH1 on the expression of cyclin D1, caspase-3, matrix metalloproteinase (MMP)-9 were observed by western blot. Our results showed that the expression rate of SASH1 mRNA in osteosarcoma tissues was significantly lower than that in normal bone tissue (p = 0.000), that the expression rate of SASH1 mRNA in the carcinoma tissues from patients with lung metastasis was significantly lower than that from patients without lung metastasis (p = 0.041), and that the expression rate of SASH1 mRNA also decreased with increasing Enneking stage (p = 0.032). However, the mRNA expression of SASH1 in osteosarcoma was independent of the patient's gender, age, and tumor size (p = 0.983, 0.343, 0.517, respectively). The SASH1 protein displayed a down-regulation in osteosarcoma tissues compared to normal bone tissue (p = 0.000), displayed a down-regulation in osteosarcoma tissues from patients with lung metastasis compared to from patients without lung metastasis (p = 0.000), and displayed a gradual decrease with increasing Enneking stage (p = 0.000). In addition, the MG-63 cells from pcDNA3.1-SASH1 group exhibited significantly reduced cell viability, proliferation, and invasive ability compared to the empty vector group and blank control group (p = 0.023, 0.001, respectively), and there was no difference between the empty vector group and blank control group. The pcDNA3.1-SASH1 group displayed significantly more apoptotic cells than the empty vector group and blank control group (p = 0.004). The expression of cyclin D1, MMP-9 displayed a down-regulation in MG-63 cells from pcDNA3.1-SASH1 group compared to the empty vector group and blank control group (p = 0.000, 0.001, respectively) and the expression levels of caspase-3 displayed an up-regulation in MG-63 cells from pcDNA3.1-SASH1 group compared to the empty vector group and blank control group (p = 0.000). Taken together, these data indicated that the overexpression of SASH1 might be associated with the inhibition of growth, proliferation, and invasion of MG-63 cells and the promotion of apoptosis of MG-63 cells.

TRAF6 regulates proliferation, apoptosis, and invasion of osteosarcoma cell.

TRAF6, a unique tumor necrosis factor receptor-associated factor (TRAF) family member, possesses a unique receptor-binding specificity that results in its crucial role as the signaling mediator for TNF receptor superfamily and interleukin-1 receptor/Toll-like receptor superfamily. TRAF6 plays an important role in tumorigenesis, invasion and metastasis. This study aimed to explore the expression of TRAF6 in osteosarcoma tissues and its correlation to the clinical pathology of osteosarcoma and to discuss the relationship between TRAF6 expression and osteosarcoma invasion. These data will provide the experimental base for the biological treatment of osteosarcoma in the future. Using RT-PCR and Western blot, the results showed that the expression rate of TRAF6 mRNA in osteosarcoma tissues was significantly higher than that in normal bone tissue (p < 0.05), that the expression rate of TRAF6 mRNA in the carcinoma tissues from patients with lung metastasis was significantly higher than that from patients without lung metastasis (p < 0.05), and that the expression rate of TRAF6 mRNA also increased with increasing Enneking stage (p < 0.05). However, the mRNA expression of TRAF6 in osteosarcoma was independent of the patient's gender, age, and tumor size (p > 0.05). The TRAF6 protein displayed an up-regulation in osteosarcoma tissues compared to normal bone tissue (p < 0.05), displayed an up-regulation in osteosarcoma tissues from patients with lung metastasis compared to from patients without lung metastasis (p < 0.05), and displayed a gradual increase with increasing Enneking stage (p < 0.05). By the technique of RNA interference, the expression of TRAF6 in the human osteosarcoma MG-63 cell line was down-regulated, and the invasive ability of MG-63 cells was examined. The results showed that TRAF6 protein expression was significantly decreased in the MG-63 cells from TRAF6 siRNA-transfected group (p < 0.05), and the proliferation ability of MG-63 cells and the number of MG-63 cells that passed through the Transwell chamber were significantly lower than that in the non-transfected control group as well as the transfected control group (p < 0.05). In addition, the percentage of MG-63 cells undergoing apoptosis was significantly higher in the TRAF6 siRNA-transfected group compared with the non-transfected control group as well as the transfected control group (p < 0.05). The expression of p-p65, cyclin D1, MMP-9 was down-regulated in the MG-63 cells from TRAF6 siRNA-transfected group. The expression of caspase 3 was up-regulated in the MG-63 cells from TRAF6 siRNA-transfected group compared to the non-transfected control group as well as the transfected control group (p < 0.05). To make a long story short, the overexpression of TRAF6 in osteosarcoma might be related to the tumorigenesis, invasion of osteosarcoma.

Regulation of inflammatory response in human chondrocytes by lentiviral mediated RNA interference against S100A10.

OBJECTIVE: The aim of the present study was to evaluate the effects of S100A10 silencing on the inflammatory response in human chondrocytes (HCs).The inflammation induced by lipopolysaccharide (LPS) was investigated in HCs in which the S100A10 was blocked with a lentiviral shRNA vector. METHODS: A lentiviral shRNA vector targeting S100A10 was constructed and packaged to effectively block S100A10 expression in HCs. HCs were infected with the lentivirus. S100A10 expression levels in HCs were detected by western blot analysis. Enzyme-linked immunosorbent assay (ELISA) was employed to evaluate the change of cytokine secretion levels. The effects of S100A10 silencing on the activation of mitogen-activated protein kinases (MAPKs) and NF-κB signaling pathway were also determined by western blot analysis. In addition, fluo-3-AM was used to demonstrate the change in calcium mobilization. RESULTS: Lentivirus effectively infected the HCs and inhibited the expression of S100A10. HCs with downregulated S100A10 showed significantly decreased production of inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-10. S100A10 silencing markedly suppressed the activation of MAPKs induced by LPS. Furthermore, the calcium concentration increase in HCs stimulated by LPS was also inhibited by S100A10 knockdown. CONCLUSION: Our investigation demonstrated that S100A10 might be considered as a potential target for anti-inflammatory treatment.