The squamous cell carcinoma cell line NOKT-1 was successfully established from the right tongue of a 74-yearold Japanese man. Pathological diagnosis of the original tumor was moderately differentiated squamous cell carcinoma. NOKT-1 cells were transplanted subcutaneously into nude mice and xenograft was formed. In addition, the NOKT-1-XG cell line was established from the transplanted tumor of NOKT-1 cells. NOKT-1 cells and NOKT-1-XG cells were epithelial neoplastic and pleomorphic cells, which were similar. Immunocytochemistry revealed that NOKT-1 and NOKT-1-XG cells were CK17 and human mitochondria positive. To authenticate the NOKT-1 cell line and NOKT-1-XG cell line, we examined cross-contamination with other cell lines using short tandem repeat analysis, the results of which showed that NOKT-1 and NOKT-1-XG are new cell lines. Four of the 16 loci, corresponding to 25%, were different between these two cell lines, which indicates that the NOKT-1 genome was altered by transplantation. Moreover, in AM, NOKT-1 did not have a Y chromosome, whereas NOKT-1-XG had. Despite the genetic differences, a collagen gel droplet-embedded culture drug susceptibility test demonstrated that NOKT-1 cells derived from the original tumor and the NOKT-1-XG cell line had the same sensitivity. This cell line could be very useful for the development of immunotherapy and chemotherapy regimens and research on cancer etiology.
Propofol is an intravenous anesthetic and used for sedation and general anesthesia in a treatment involving invasion of the bone. Despite the period and dose of propofol being varied depending on the purpose of administration, effects of propofol on bone metabolism are less investigated. Osteoclast progenitor cells fuse with each other to differentiate into osteoclasts in the presence of receptor activator of nuclear factor kappa B (RANK) ligand (RANKL), and osteoclasts play a central role in bone resorption during bone remodeling. In the current study, we examined the effects of both temporary and continuous propofol stimulation on RANKL-induced osteoclastogenesis. RAW264.7 cells were stimulated with 0, 10, 20, or 30 µM propofol for 5 h, 1.5 days, or 4 days in the presence of RANKL. At the end of stimulation for 5 h and 1.5 days, cells were continued to be cultured in medium containing RANKL without propofol until the end of total culture period. The expression of dendritic cell-specific transmembrane protein (DC-STAMP) and osteoclast stimulatory transmembrane protein (OC-STAMP) that are involved in the fuse of cells as well as the expression of RANK and leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4), both are receptor of RANKL but have oppose effects to osteoclastogenesis are examined by real-time PCR. The formation of osteoclast-like cells was verified by tartrate-resistant acid phosphatase staining. Propofol stimulation for 1.5 or 4 days suppressed the expression of DC-STAMP, OC-STAMP and RANK, as well as the formation of osteoclast-like cells, whereas the expression of LGR 4 was increased by propofol stimulation for 4 days. These findings suggest that several hours of propofol administration do not affect RANKL-induced osteoclastogenesis. However, several days of propofol exposure may suppress the differentiation of osteoclasts due to decreased expression of DC-STAMP, OC-STAMP and RANK, as well as increased expression of LGR4.
In this study, we determined whether cisplatin can induce epithelial-mesenchymal transition (EMT) via the activation of Sonic hedgehog (Shh) or glioma-associated antigen-1 (Gli1) signaling pathway in mouse Hertwig’s epithelial root sheath (HERS) cells using a genetic knockdown approach. HERS cells treated with a low concentration of cisplatin (0.5 µM) for 24 h showed no reduction in the cell viability; however, there was a significant increase in the percentages of nuclear staining with γH2AX as compared to that with untreated control cells, indicating that 0.5 µM cisplatin induces DNA damage. Further, 0.5-µM cisplatin-treated cells provided an induction of EMT, showing decreased and increased expression of epithelial and mesenchymal markers, respectively. Enhancement in the EMT activity in cisplatin-treated HERS cells was correlated with increased expression of Shh and accelerated translocation and accumulation of Gli1 expression into the nucleus. The RNA interference-mediated silencing of Gli1 suppressed the acceleration of EMT in cisplatin-treated HERS cells; this was confirmed by no down-regulation or up-regelation in the expression of E-cadherin and vimentin, respectively, along with no increased expression of Snail expression. These findings suggest that the activation of Shh/Gli1 signaling pathway may be required for the enhancement of EMT in cisplatin-treated HERS cells.
We evaluated the effects of zoledronic acid (ZOL) on human gingival fibroblasts (HGFs) and human umbilical vein endothelial cells (HUVECs) associated with wound healing in oral soft tissues. HGFs and HUVECs were divided into two groups: a culture media control group and a group exposed to ZOL (50 μM). Cell proliferation was measured after 2, 4, 6, and 8 days. The migration ability of cells was measured for each experiment using the wound healing assay. The apoptosis rate was confirmed using the apoptosis assay. Culture supernatants were collected from each experimental group and vascular endothelial growth factor (VEGF) production in the culture media was measured using enzyme-linked immunosorbent assay (ELISA). Further, the expression level of VEGF-A was evaluated and compared using real-time quantitative polymerase chain reaction. The proliferation and migration abilities of both HGFs and HUVECs were confirmed to be suppressed by the addition of ZOL, resulting in apoptosis. ELISA revealed that the quantity of VEGF produced in HGFs was significantly higher in the ZOL group than in the control group until 2 days after the addition of ZOL. In HGFs, the mRNA expression levels of intracellular VEGF-A increased with the addition of ZOL, demonstrating the production of VEGF. In contrast, in HUVECs, although the mRNA expression levels of endogenous VEGF-A increased with the addition of ZOL, VEGF production was considerably decreased in the culture supernatant, indicating the possibility of abnormalities in the autocrine functions of endogenous VEGF or intracellular signal transduction of exogenous VEGF. These data suggest the utility of therapeutic approaches directed toward abnormalities in VEGF intracellular signaling to improve medication-related osteonecrosis of the jaw soft-tissue healing.
In recent years, atmospheric pressure plasma jet (APPJ) has been widely developed for various medical applications, such as medical equipment sterilization, gene transfection, cell proliferation, and wound healing. In particular, non-thermal APPJ enables direct treatment of the biological system without any thermal-associated damage. The effect of cells on APPJ depends upon the gas species used in the treatment. However, the mechanisms underlying osteoblast differentiation mediated by APPJ with nitrogen are yet to be studied. This study investigated the effects of nitrogen-APPJ on osteoblast differentiation by assessing the transcription factors, extracellular matrix proteins (ECMPs), alkaline phosphatase (ALP) activity, and the mRNA and protein expressions of ALP, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), in an osteoblast mouse cell line. We found that nitrogen-APPJ induced osteoblast differentiation-related transcription factors (runt-related transcription factor 2 [Runx2] and osterix), osteocalcin (OCN), and ALP activity, as well as reduced the mRNA and protein expressions of iNOS and COX-2. Thus, we concluded that APPJ affects differentiation of the osteoblast cells.
Osteoarthritis (OA) is a chronic degenerative joint disease with a multifactorial etiology including inflammatory mediators. The effects of vascular endothelial growth factor (VEGF) on OA have been studied widely in the field of orthopedics. This study aimed to evaluate whether VEGF could affect the progression of OA in the mouse temporomandibular joint (TMJ). C57BL/6J mice (n = 54) were assigned to three groups, namely, the VEGF+Discectomy, Discectomy, and Sham groups. OA was induced with a discectomy performed on the TMJ in 12-week-old mice in the VEGF+Discectomy and Discectomy groups. Mice in the VEGF+Discectomy group underwent intra-articular VEGF administration after discectomy. For the mice of the Sham group, the joint space was opened surgically, but the disc was not removed. At 4, 8, and 16 weeks after the induction of TMJ OA, the animals were sacrificed. Condylar dimensions and cartilage thickness were measured. Histological changes of the cartilage were assessed using a modified Mankin scoring system. The VEGF+Discectomy group showed a marked reduction of cartilage thickness at 16 weeks post-surgery. According to the modified Mankin scoring system, the VEGF+Discectomy group exhibited the highest scores for the severe reduction of safranin O staining, hypocellularity, and clefts in deep cartilage zones at 16 weeks post-surgery. In the surgically induced TMJ OA mouse model, the VEGF+Discectomy group exhibited highly progressive OA changes in articular cartilage. The detrimental effects of VEGF on TMJ OA may be via its role in the promotion of degradation.
We aimed to detect the expressions of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) in different development stages of mandibular first molar and incisor in BALB/c mice, and to uncover the action mechanisms of YAP and TAZ during tooth development. BALB/c mice were randomly divided into 5 groups, i.e. 19.5-d-old embryo, and 0-, 6-, 14- and 28-d-old mice (n=8). To detect the expression changes of YAP and TAZ during tooth development, the mandibular tissues of first molar and incisor were collected and subjected to HE staining and SABC immunohistochemical staining. YAP expression was observed in the molar gem of 19.5-d-old embryo, which increased gradually in mice on 0, 6, 14 and 28 d after birth. TAZ was expressed in granule shape in the molar germ and tooth of 19.5-d-old embryo and 0-d-old mice. Expression of TAZ was observed on 6, 14 and 28 d after birth, which was the lowest on 6 d after birth and rose on 14 and 28 d. YAP and TAZ are specifically expressed and localized during the development of mandibular first molars of mice, probably being involved in ameloblast and odontoblast differentiation and dentin calcification.
The conventional culture method of human induced pluripotent stem cells (hiPSCs) has been performed in colony cultures using feeder cells, such as mouse embryonic fibroblasts, which require setup times and procedural complexity, a potential risk of transmission of animal pathogens. Besides, the colony culture exhibits slow growth rate and often give rise to heterogeneous cellular states. However, developing technical methodologies of hiPSCs remains pivotal for use in medical applications and research. Here, we investigated whether hiPSCs passaged and expanded as single cells under feeder-free conditions could differentiate into ectodermal-epithelial cells as a source of cells for future regenerative medicine research. First, an hiPSC line 253G1 was cultured in colonies maintained on feeders and subsequently transferred to a single cell on feeder-free. hiPSCs were then cultured as single cells for 28 days in an induction medium supplemented with retinoic acid, bone morphogenetic protein 4, and N2 supplement for epithelial cell differentiation. The expression of epithelial markers, tumor protein p63 (P63), cytokeratin (CK) 18, and CK14 in induced cells was evaluated over time using real-time polymerase chain reaction, western blotting, and immunocytochemistry. Results showed that hiPSCs cultured as single cells expressed pluripotency markers, as evidenced by colony cultures maintained on feeders. On day 7 post-induction, hiPSCs assumed a cobblestone-like morphology in the epithelial induction medium. Induced cells displayed increased mRNA expression levels of CK18, P63, and CK14 during the 28-day induction period. Furthermore, the expression levels of CK18, P63, and CK14 were detected via western blotting and immunocytochemistry. Our findings suggest that hiPSCs cultured as single cells could be differentiated into epithelial cells.
Most studies of artificial intelligence in the medical field involve classification problems, but few consider recognition of one characteristic point in images or regression analysis such as data recognition. In this research, we constructed a fundamental convolutional neural network framework for regression analysis. Images of the handwritten digit “3” from the MNIST dataset were used as training data, with the protruding middle point as an image feature point. Input images and training data (x1, y1) were connected to 6 convolutional layers and then run through 2 affine layers to produce the output data (x2, y2). The loss function was the mean radial error (MRE) between the training and output data. After machine learning, the error converged to 0.75 pixels on average. We expect that this algorithm can be clinically applied to points having certain characteristics in images, such as locating hard tissue lesions or recognizing measurement points in cephalograms.
Titanium is most widely used for implants. Almost all the histological studies of the implants have been carried out with polished specimens and the use of frozen sections are challenging. This study focused to define the formation process of bone around titanium with frozen sections made from tissue implanted with a commercially pure titanium foil (Ti-foil). We inserted the Ti-foil with a 30-μm thickness into rat femurs and extracted it after 1, 3, 5, 7, 10, 15, and 30 days. After freezing, they were sliced into 3-μm thick serial frozen sections and the sections were used for hematoxylin and eosin (H&E) staining, Masson trichrome staining, Alizarin Red S staining, enzymatic staining (ALPase and TRAPase), immunostaining (osteopontin, osteocalcin, and collagen type I), and elemental analysis. At 1 day after the implantation, the area around the Ti-foil was filled with blood and there are no collagen fibers and no proliferated cells in the area. At 3 days, the new collagen fibers were observed on the marrow side of the blood clot. The proliferated cells were observed around the fibers and they were positive for immunostaining to osteopontin, osteocalcin, and collagen type I. After 5 days, calcium precipitation was observed on the newly formed collagen fibers. After 7 days, the calcification progressed and started to reach the Ti-foil surface. After 10 days, calcification progressed to the area around the Ti-foil. At 15 days, a remarkable bone resorption appears on the marrow side. At 30 days, further resorption was observed and bony tissue was seen on the Ti-foil surface only. These results clearly showed that the bone around the Ti-foil is formed after undergoing the blood clotting process around the Ti-foil, osteoblast and fibroblast proliferation, calcium precipitation on the collagen fibers, bone formation around the Ti-foil, and bone resorption by osteoclasts.
This study aimed to examine whether a low concentration of etoposide can accelerate osteogenesis via the activation of peptidyl-prolyl isomerase Pin1 in human MG63 cells using a genetic knockdown approach. MG63 cells treated with 0.1 μM etoposide for 24 h showed neither reduction in cell viability nor induction of cellular senescence; however, there was a significant increase in the percentage of nuclear staining with γH2AX as compared with that in untreated control cells, indicating that 0.1 μM etoposide induces weak DNA damage response (DDR) in the cells. Treatment with 0.1 μM etoposide accelerates osteogenesis in osteogenic induction medium (OIM)-cultured MG63 cells, demonstrating increased expression of Runx2 and Osterix and intense alkaline phosphatase (ALP) staining, as compared with cells treated without etoposide. In addition to those osteogenic markers, Pin1 expression was upregulated in the etoposide-treated cells, suggesting that the weak DDR may provide an interaction between Pin1 activation and osteogenic markers. The RNA interference-mediated silencing of Pin1 suppressed the expression of osteogenic markers and ALP staining in the OIM-cultured cells pretreated with 0.1 μM etoposide. Based on these findings, we suggest that a low concentration of etoposide induces mild DDR to activate Pin1, eventually promoting OIM-induced osteogenesis in the MG63 cells.
Our objective was to perform a quantitative evaluation of changes in the micro/nanostructural characteristics of entheses in rats with reduced masticatory muscle functional pressure, with the aim of elucidating the mechanism whereby masticatory muscle functional pressure contributes to growth and development of the mandible from a biomechanical perspective. Male Wister rats aged 4, 11, 18 and 25 weeks were divided into a Botox group injected with a botulinum toxin serotype A formulation to reduce muscle function (BTX) and a control group (CTRL). They were euthanized 6 weeks later and bone quality at the masseter insertion at the mandibular was analyzed. In the BTX group, the number of fibrous chondrocytes at entheses was significantly lower than in the CTRL group at all ages. The diameter of collagen fiber bundles in rats in the BTX group injected with BoNT/A during their growth phase was significantly smaller than that of rats in the CTRL group. In the mandibles of rats in the CTRL group the preferential alignment was consistent with the orientations of the muscle and tendon, but in growing rats treated with BTX, it was less closely aligned with the orientations of the muscle and tendon.
This study aimed to investigate the success factors of the bone lid surgery technique in the maxillofacial region. A retrospective cohort study was performed on 30 maxillofacial patients who underwent bone lid surgery between January 2014 and December 2019 at our hospital. The predictor variables consisted of clinical factors that were classified as attribute (age and sex), health status (smoking and alcohol intake), anatomical (maxillary/mandibular site, left/right side, and cortical bone thickness), lesion (lesion size, location, and pathological diagnosis), and treatment variables (differences in absorbable osteosynthesis materials). The outcome variable was the incidence of bone lid necrosis after surgery. Various risk factors for postoperative bone lid necrosis were investigated statistically. A 𝑝 value <0.05 was considered statistically significant. Postoperative bone lid necrosis was observed in three patients (10.0%). No significant differences in the attribute, anatomical, and treatment status variables were noted. Significant differences were observed between smoking (p=0.005) and alcohol intake (p=0.003) in the health status variables. There was a significant difference in the distance of the lesion from the alveolar bone crest in the lesion variables (p=0.037). Smoking and alcohol consumption were the health status variables found to be risk factors for bone lid necrosis. In addition, proximity to the alveolar crest was also a risk factor for lesion development.
The objective of this study is to investigate the influence of high cholesterol condition on apical periodontitis and bone resorption in rats. Six-week-old rats were fed a high cholesterol diet (HCD) and compared to normal diet (ND) rats. The pulp of the first maxillary molar was exposed and apical periodontitis was generated experimentally, and bone resorption was evaluated. At 6 weeks after pulp exposure, apical periodontitis were noted histologically and radial bone resorption was observed in the HCD rats. TRAP staining revealed the number of osteoclasts in the HCD group to be significantly higher than that in ND rats (p<0.05). Significant differences in mRNA expression in toll-like receptor 2 (TLR2) and RANKL were detected in HCD rats. These results suggest that a high-cholesterol condition promotes apical periodontitis and bone resorption via expression of TLR2 and RANKL in rats.
To discuss the characteristics of Suicidal Jumper Fractures (SJF) and evaluate clinical outcomes treated with lumbopelvic fixation. From August 2007 to August 2012, nine consecutive cases with SJF were included into the study. The clinical data of these cases including fracture classifications, associated injuries and the degrees of neurological impairment were analyzed and assessed preoperatively. All cases were followed-up continuously after an average of 42 ± 4.54 months (range: 34-90 months). All fractures healed after 5 ± 1.53 months (range: 3-11 months). None of these cases had fracture re-displacement and fixation failure. Based on the Majeed scoring system, the postoperative prognosis of these patients were excellent in four cases, good in three cases, fair in one case, and poor in one case. There was a significant improvement in neurologic deficiency in all postoperative patients, and their average Gibbons scores changed from 3.12 ± 0.23 preoperatively to 1.54 ± 0.45 postoperatively; and the difference was statistically significant (t=3.22, P<0.05). Lumbopelvic fixation has a significant advantage in the treatment of this series of fractures, and can help obtain a satisfactory clinical outcome. Intraoperative nerve decompression is necessary if indications exist and the improvement of neurological impairment is optimistic.
Muscles of mastication are replaced by fibrous tissue in oral submucous fibrosis (OSMF). Muscular changes, in the form of dystrophy because of muscular over activity is observed in most of the cases of OSMF. One hundred OSMF patients and 25 healthy individuals were included in this study. The patients were categorized into four groups on the basis of mouth opening. For every individual, age, sex, weight, height and body mass index (BMI) were documented. Burning sensation, mouth opening, maximum bite force (MBF) was evaluated among study groups and control group. The OSMF individuals were injected with hyaluronidase 1500 IU mixed in 1.5 ml of dexamethasone and 0.5 ml of lignocaine HCL intralesionally twice a week for one month and control subjects were given placebo capsules and all the parameters were revaluated. Statistical analysis was carried out using Student’s independent t-test, Analysis of variance and Tukey’s post hoc test. No significant difference was observed in mean age, mean height, weight, BMI and the presence of the number of intact teeth between controls and OSMF individuals. A significant decrease in anterior MBF in group III and IV and posterior MBF of both sides in groups II, III and IV was noted. After treatment there was a significant improvement in anterior MBF in group III and posterior MBF in groups II and III OSMF patients. It was concluded that, MBF was reduced in patients with OSMF and it is improved in early and moderate cases after intralesional hyaluronidase and corticosteroid therapy.