Effect of α-Mangostin on Interleukin-11 and Runt-related Transcription Factor-2 Gene Expression on Cell Line Osteoblast Cultures Induced with Lipopolysaccharide.

Background: Loss of permanent teeth after tooth extraction without replacement of missing teeth can result in impaired masticatory, esthetic, phonetic functions, and impaired balance of the masticatory organ in the mouth. Therefore, a method is needed to inhibit the alveolar bone resorption process so that the dimensions of the tooth socket can be maintained vertically or horizontally until the time of implant placement, which is called the socket preservation procedure. α-mangostin is known to have a potential anti-inflammatory effect and most likely can be used as a potential therapeutic agent to inhibit bone resorption caused by posttooth extraction inflammatory processes. Aims: The aim of the study was to determine the effect on the inflammatory process and osteogenesis on osteoblast cell line culture by induction with lipopolysaccharide (LPS) and α-mangostin. Materials and Methods: This was an in vitro laboratory experimental study on mouse osteoblast cell line culture. The treatment was given with LPS, α-mangostin, and combination on osteogenic medium, using the same concentration for all concentrates. The sample will then be processed and analyzed using the real-time polymerase chain reaction. Results: The highest interleukin-11 (IL-11) gene expression was found in α-mangostin treatment, but there was no significant difference in IL-11 expression between the study groups. The highest runt-related transcription factor-2 (RUNX-2) gene expression was found in a group that received induction with LPS and α-mangostin, and from these results, it was found that there was a significant difference in RUNX-2 expression between the study groups. Conclusions: LPS and α-mangostin can increase osteogenesis in osteoblast cell culture in the osteogenic medium.

Quality of Life Evaluation of Postsurgical Mandibular Fracture Patients with Oral Health Impact Profile 14 and General Oral Health Assessment Index Parameters.

OBJECTIVE: Mandibular fracture is the most common maxillofacial fracture accompanied by complaints of malocclusion and pain. This causes a decrease in the quality of life. Mandibular fracture management can be done with open reduction and internal fixation or intermaxillary fixation. The Oral Health Impact Profile (OHIP 14) and the General Oral Health Assessment Index (GOHAI) were used to evaluate the quality of life after surgical treatment based on the distribution of age, sex, type of neglect, and surgical management. MATERIALS AND METHODS: This research is an analytic study with an analytical observational method with total sampling. The total sample used was 15 patients during the 2006 to 2020 period. The results of this study were scored, and then, the data were processed using the eta test. RESULTS: The results of the study based on the OHIP 14 parameters showed the results of each distribution, namely, age: p = 0.154, gender: p = 0.080, neglected type: p = 0.080, and management: p = 0.419. Meanwhile, the GOHAI parameters showed the results of each distribution, namely, age: p = 0.105, gender: p = 0.356, neglected type: p = 0.356, and management p = 0.286. The results of this distribution showed that there was no significant difference between patients' quality of life based on age, sex, neglected type, and treatment using both OHIP 14 and GOHAI parameters. CONCLUSIONS: The results obtained in this study using characteristics of age, gender, type of fracture, type of neglect, and management did not have a significant effect on the level of patient satisfaction after surgery, using both OHIP 14 and GOHAI questionnaires.

Osteoinductive and Osteogenic Capacity of Freeze-Dried Bovine Bone Compared to Deproteinized Bovine Bone Mineral Scaffold in Human Umbilical Cord Mesenchymal Stem Cell Culture: An In Vitro Study.

OBJECTIVE: Freeze-dried bovine bone scaffold (FDBB) or decellularized FDBB (dc-FDBB) was developed as an ideal scaffold with osteoinductive properties. This research aims to compare the osteoinductive properties marked by the expression of runt-related transcription factor-2 (RUNX2) and Osterix (OSX) and the osteogenic capacity of these scaffolds imbued with human umbilical cord mesenchymal stem cells (hUCMSCs). MATERIALS AND METHODS: This study was performed in five experimental groups: a negative control group (C-) of hUCMSCs with a normal growth medium, a positive control group (C + ) of hUCMSCs with an osteogenic medium, experimental group 1 (E1) with an FDBB conditioned medium (CM), and experimental group 2 (E2) with a dc-FDBB-CM, and a third experimental group (E3) consisting of a DBBM-CM. Alizarin red staining was performed to qualitatively assess osteoinductive capacity. RUNX2 and OSX expression was quantified using real-time quantification polymerase chain reaction with two replications on day six (D6) and day 12 (D12) as fold changes. RESULTS: This experiment revealed that hUCMSCs were positively expressed by CD73, CD90, and CD105 but were not expressed by CD34. Alizarin red staining showed that E1 had the most calcium deposition on D6 and D12, followed by E3 and then E2 The RUNX2 and OSX expression was higher in E1 but this difference was not significant. The OSX expression in E1,E2,E3 was lower on D12 and C+ of OSX had the highest expression. There was a significant difference of fold change measured between all groups (p < 0.05), and there was no significant difference between any of the groups treated with OSX and RUNX2 on D6 and D12. CONCLUSION: FDBB osteoinduction and osteogenic capacity were higher when compared with DBBM and dc-FDBB.

Melatonin­induced miR­181c­5p enhances osteogenic differentiation and mineralization of human jawbone­derived osteoblastic cells.

Our previous study revealed that treatment with a combination of fibroblast growth factor­2 and melatonin (MEL) synergistically augmented osteogenic activity and mineralization of MC3T3­E1 mouse preosteoblast cells. Thus, the objective of the present study was to assess the effect of MEL on osteogenetic characteristics in human osteoblastic cells. Human jawbone­derived osteoblastic (hOB) cells were isolated from mandibular bone fragments. RUNX family transcription factor 2 (Runx2) expression, alkaline phosphatase (ALP) enzyme activity and the mineralization ability of hOB cells in the presence of MEL were evaluated. Microarray analysis was also performed to assess the expression of MEL­induced microRNAs (miRNAs/miRs) in hOB cells. Treatment with MEL significantly enhanced Runx2 expression, ALP activity and mineralization staining. However, this effect was significantly reduced following transforming growth factor­ß1 treatment. In total, 124 miRNAs were differentially expressed in MEL­treated hOB cells, compared with untreated cells. Of the upregulated miRNAs, miR­181c­5p exhibited the largest fold change. Runx2 mRNA expression and mineralization staining in the presence of MEL were significantly reduced following transfection with a miR­181c­5p inhibitor. In addition, transfection with miR-181c-5p mimics significantly increased Runx2 expression and mineralization staining. These results suggested that MEL­induced miR­181c­5p was involved in osteogenic differentiation and mineralization of hOB cells. Using TargetScan, a putative miR­181c­5p binding site was identified in the Notch2 gene. Moreover, Notch2 mRNA and protein expression levels in hOB cells were significantly reduced following transfection with miR­181c­5p mimics, confirming Notch2 as a target gene for miR­181c­5p. Notch2 siRNA knockdown significantly increased Runx2 expression and mineralization staining, which suggested that Notch2 may negatively regulate osteogenic differentiation of hOB cells by downregulating Runx2. In conclusion, MEL­induced expression of miR­181c­5p enhanced osteogenic differentiation and calcification of hOB cells.

CD44high/ESAlow squamous cell carcinoma cell-derived prostaglandin E2 confers resistance to 5-fluorouracil-induced apoptosis in CD44high/ESAhigh cells.

We previously found that CD44high/ESAlow head and neck squamous cell carcinoma (HNSCC) cells harboring high dihydropyrimidine dehydrogenase (DPD) expression exhibited potent resistance to 5-fluorouracil (5-FU)-induced apoptosis. In addition, susceptibility of HNSCC cells to 5-FU was compromised in the presence of cyclooxygenase 2 (COX2)-derived prostaglandin E2 (PGE2). In this study, we examined 5-FU-induced apoptosis in sorted cell populations (i.e., CD44high/ESAlow, CD44high/ESAhigh, and CD44low cells from the HNSCC cell line A-253) to clarify the anti-apoptotic effect of PGE2 on CD44high cells. Notably, CD44high/ESAlow cells upregulated PGE2, compared with other populations. To investigate the effect of CD44high/ESAlow cell-derived PGE2 on CD44high/ESAhigh cells, direct and indirect co-culture assays were performed. The percentage of apoptotic cells in a culture of CD44high/ESAhigh cells was significantly reduced when they were directly and indirectly co-cultured with CD44high/ESAlow cells. Furthermore, 5-FU-induced apoptosis of CD44high/ESAhigh cells was significantly increased in the presence of an inhibitor of the PGE2 receptors (EP1/EP2) when CD44high/ESAhigh cells were co-cultured with CD44high/ESAlow cells. These results suggest that CD44high/ESAlow cell-derived PGE2 may contribute to the inhibition of 5-FU-induced apoptosis in CD44high/ESAhigh cells. Additionally, NR4A2 knockdown enhances 5-FU-induced apoptosis in CD44high/ESAhigh cells, suggesting that PGE2 attenuates 5-FU-induced apoptosis in an NR4A2-dependent manner in CD44high/ESAhigh cells. In conclusion, CD44high/ESAlow cells contribute to induction of resistance to 5-FU in CD44high/ESAhigh cells through provision of PGE2. CD44high/ESAlow cell-targeted therapy may be effective in treatment of HNSCC.

Preoperative oral health care reduces postoperative inflammation and complications in oral cancer patients.

The records of 70 patients with oral cancer who were treated at a single institution between 2008 and 2014 were reviewed. The body temperature, white blood cell count, and C-reactive protein (CRP) levels were compared between those who had received preoperative oral care (oral care group) and those who had not received any (non-oral care group). When the patients were divided into those who underwent minimally invasive surgery and those who underwent severely invasive surgery, the mean CRP level in the early postoperative period was lower in the oral care group as compared with the non-oral care group in those who underwent minimally invasive surgery as well as those who underwent severely invasive surgery. However, the mean CRP level was most evidently reduced in the severely invasive group on days 1 and 3-5. However, no significant differences were observed with regard to the percentage of postoperative infectious complications (for example, surgical site infection, anastomotic leak and pneumonia) between the oral care (13.6%) and non-oral care (20.8%) groups, though a reduced prevalence of postoperative complications following preoperative oral care was noted. The results of the present study suggest that preoperative oral care can decrease inflammation during the early postoperative stage in patients with oral cancer who undergo severely invasive surgery.

Higher prevalence and gene amplification of HPV16 in oropharynx as compared to oral cavity.

OBJECTIVE: The objective of this study was to clarify differences regarding HPV16 infection and gene amplification between the oral cavity and oropharynx in healthy individuals. MATERIAL AND METHODS: The subjects were 94 healthy asymptomatic individuals (41 males, 53 females; mean age 58.6 years, range 16-97 years) who visited the Department of Oral and Maxillofacial Reconstructive Surgery of the Hiroshima University Hospital from 2014 to 2015. Oral epithelial cells were collected from oral rinse and pharynx gargle samples and placed in saline. The human endogenous retrovirus gene ERV3-1 was used as a reference to estimate the number of human cells in each sample. DNA samples were extracted from approximately 10,000 human cells and tested for HPV16 DNA by PCR using a type-specific primer. Similarly, we analyzed the HPV16 viral copy number in HPV16-positive cases using real-time PCR to examine genomic amplification. RESULTS: The percentage of HPV16-positive cases was higher in the gargle (28.7%) as compared to the rinse (16.0%) samples. In the oral rinse samples, males (26.8%) showed a significantly higher rate of HPV16 than females (7.5%) (P=0.021). Importantly, in older subjects (aged 60-89 years), gargle samples showed a significantly higher rate of HPV16 (33.3%) than oral rinse samples (13.7%) (P=0.034). The average number of viral copies was approximately 8 times higher in the gargle than in the oral rinse samples (0.16±0.27 vs. 1.35±1.26 copy numbers per cell), a significant difference (P<0.001). CONCLUSION: Our findings suggest that the oropharynx is more susceptible to HPV16 infection as compared to the oral cavity, while HPV16 gene amplification is also more commonly found in the oropharynx.

Higher prevalence and gene amplification of HPV16 in oropharynx as compared to oral cavity

ABSTRACT Objective The objective of this study was to clarify differences regarding HPV16 infection and gene amplification between the oral cavity and oropharynx in healthy individuals. Material and Methods The subjects were 94 healthy asymptomatic individuals (41 males, 53 females; mean age 58.6 years, range 16-97 years) who visited the Department of Oral and Maxillofacial Reconstructive Surgery of the Hiroshima University Hospital from 2014 to 2015. Oral epithelial cells were collected from oral rinse and pharynx gargle samples and placed in saline. The human endogenous retrovirus gene ERV3-1 was used as a reference to estimate the number of human cells in each sample. DNA samples were extracted from approximately 10,000 human cells and tested for HPV16 DNA by PCR using a type-specific primer. Similarly, we analyzed the HPV16 viral copy number in HPV16-positive cases using real-time PCR to examine genomic amplification. Results The percentage of HPV16-positive cases was higher in the gargle (28.7%) as compared to the rinse (16.0%) samples. In the oral rinse samples, males (26.8%) showed a significantly higher rate of HPV16 than females (7.5%) (P=0.021). Importantly, in older subjects (aged 60-89 years), gargle samples showed a significantly higher rate of HPV16 (33.3%) than oral rinse samples (13.7%) (P=0.034). The average number of viral copies was approximately 8 times higher in the gargle than in the oral rinse samples (0.16±0.27 vs. 1.35±1.26 copy numbers per cell), a significant difference (P<0.001). Conclusion Our findings suggest that the oropharynx is more susceptible to HPV16 infection as compared to the oral cavity, while HPV16 gene amplification is also more commonly found in the oropharynx.

Combined effects of melatonin and FGF-2 on mouse preosteoblast behavior within interconnected porous hydroxyapatite ceramics - in vitro analysis.

Objective Biocompatible materials such as interconnected porous hydroxyapatite ceramics (IP-CHA) loaded with osteogenic cells and bioactive agents are part of an evolving concept for overcoming craniofacial defects by use of artificial bone tissue regeneration. Amongst the bioactive agents, melatonin (MEL) and basic fibroblast growth factor (FGF-2) have been independently reported to induce osteoblastic activity. The present in vitro study was undertaken to examine the relationship between these two bioactive agents and their combinatory effects on osteoblastic activity and mineralization in vitro. Material and Methods Mouse preosteoblast cells (MC3T3-E1) were seeded and cultured within cylindrical type of IP-CHA block (ø 4x7 mm) by vacuum-assisted method. The IP-CHA/MC3T3 composites were subjected to FGF-2 and/or MEL. The proliferation assay, alkaline phosphatase enzyme activity (ALP), mRNA expressions of late bone markers, namely Osteocalcin (OCN) and Osteopontin (OPN), and Alizarin Red staining were examined over a period of 7 days. Results FGF-2 mainly enhanced the proliferation of MC3T3-E1 cells within the IP-CHA constructs. MEL mainly induced the mRNA expression of late bone markers (OCN and OPN) and showed increased ALP activity of MC3T3 cells cultured within IP-CHA construct. Moreover, the combination of FGF-2 and MEL showed increased osteogenic activity within the IP-CHA construct in terms of cell proliferation, upregulated expressions of OCN and OPN, increased ALP activity and mineralization with Alizarin Red. The synergy of the proliferative potential of FGF-2 and the differentiation potential of MEL showed increased osteogenic activity in MC3T3-E1 cells cultured within IP-CHA constructs. Conclusion These findings indicate that the combination of FGF-2 and MEL may be utilized with biocompatible materials to attain augmented osteogenic activity and mineralization.

Combined effects of melatonin and FGF-2 on mouse preosteoblast behavior within interconnected porous hydroxyapatite ceramics - in vitro analysis

ABSTRACT Objective Biocompatible materials such as interconnected porous hydroxyapatite ceramics (IP-CHA) loaded with osteogenic cells and bioactive agents are part of an evolving concept for overcoming craniofacial defects by use of artificial bone tissue regeneration. Amongst the bioactive agents, melatonin (MEL) and basic fibroblast growth factor (FGF-2) have been independently reported to induce osteoblastic activity. The present in vitro study was undertaken to examine the relationship between these two bioactive agents and their combinatory effects on osteoblastic activity and mineralization in vitro. Material and Methods Mouse preosteoblast cells (MC3T3-E1) were seeded and cultured within cylindrical type of IP-CHA block (ø 4x7 mm) by vacuum-assisted method. The IP-CHA/MC3T3 composites were subjected to FGF-2 and/or MEL. The proliferation assay, alkaline phosphatase enzyme activity (ALP), mRNA expressions of late bone markers, namely Osteocalcin (OCN) and Osteopontin (OPN), and Alizarin Red staining were examined over a period of 7 days. Results FGF-2 mainly enhanced the proliferation of MC3T3-E1 cells within the IP-CHA constructs. MEL mainly induced the mRNA expression of late bone markers (OCN and OPN) and showed increased ALP activity of MC3T3 cells cultured within IP-CHA construct. Moreover, the combination of FGF-2 and MEL showed increased osteogenic activity within the IP-CHA construct in terms of cell proliferation, upregulated expressions of OCN and OPN, increased ALP activity and mineralization with Alizarin Red. The synergy of the proliferative potential of FGF-2 and the differentiation potential of MEL showed increased osteogenic activity in MC3T3-E1 cells cultured within IP-CHA constructs. Conclusion These findings indicate that the combination of FGF-2 and MEL may be utilized with biocompatible materials to attain augmented osteogenic activity and mineralization.

Professional oral health care reduces the duration of hospital stay in patients undergoing orthognathic surgery.

The present study reviewed the records of 58 patients who underwent orthognathic surgery [sagittal split ramus osteotomy (SSRO), Le Fort I osteotomy, genioplasty, anterior maxillary alveolar osteotomy] between 2010 and 2015. To investigate the influence of preoperative oral health care on postoperative inflammation, infection and length of hospital stay in those patients, white blood cell (WBC) count and C-reactive protein (CRP) levels were compared between patients who received and did not receive preoperative oral care. The mean CRP level throughout the postoperative term was lower in the oral care group as compared to the non-oral care group. By contrast, the oral care group had a lower occurrence of postoperative infectious complications (surgical site infection, anastomotic leak) (13.6 vs. 20.8%) and a shorter average length of hospital stay (16.2±3.8 vs. 21.2±7.4 days). These results suggest that preoperative professional oral health care decreases the duration of hospital stay following orthognathic surgery by inhibiting inflammation and infectious complications during the postoperative stage.

Effects of Apatite Cement Containing Atelocollagen on Attachment to and Proliferation and Differentiation of MC3T3-E1 Osteoblastic Cells.

To improve the osteoconductivity of apatite cement (AC) for reconstruction of bone defects after oral maxillofacial surgery, we previously fabricated AC containing atelocollagen (AC(ate)). In the present study, we examined the initial attachment, proliferation and differentiation of mouse osteoblastic cells (MC3T3-E1 cells) on the surface of conventional AC (c-AC), AC(ate) and a plastic cell dish. The number of osteoblastic cells showing initial attachment to AC(ate) was greater than those attached to c-AC and similar to the number attached to the plastic cell wells. We also found that osteoblastic cells were well spread and increased their number on AC(ate) in comparison with c-AC and the wells without specimens, while the amount of procollagen type I carboxy-terminal peptide (PIPC) produced in osteoblastic cells after three days on AC(ate) was greater as compared to the others. There was no significant difference in regard to alkaline phosphatase (ALP) activity and osteocalcin production by osteoblastic cells among the three surface types after three and six days. However, after 12 days, ALP activity and the produced osteocalcin were greater with AC(ate). In conclusion, AC(ate) may be a useful material with high osteoconductivity for reconstruction of bone defects after oral maxillofacial surgery.

CD44(high) /ALDH1(high) head and neck squamous cell carcinoma cells exhibit mesenchymal characteristics and GSK3ß-dependent cancer stem cell properties.

BACKGROUND: CD44 and aldehyde dehydrogenase 1 (ALDH1) have been shown to be useful markers for identification of cancer stem cells (CSCs). We previously reported that glycogen synthase kinase 3ß (GSK3ß) is involved in regulation of the self-renewal ability of head and neck squamous cell carcinoma (HNSCC) CSCs. The purpose of the present study was to clarify the role of GSK3ß in CD44(high) /ALDH1(high) HNSCC cells. METHODS: Cells with greater expression of CD44 and higher ALDH1 enzymatic activity were FACS sorted from the OM-1 HNSCC cell line. The self-renewal ability of CD44(high) /ALDH1(high) cells was then examined using a tumor sphere formation assay. mRNA expressions of the stem cell markers Sox2, Oct4, and Nanog, as well as GSK3ß were evaluated by real-time RT-PCR. RESULTS: CD44(high) /ALDH1(high) cells exhibited higher tumor sphere forming ability and increased expression of stem cell markers as compared with CD44(high) /ALDH1(low) cells. Interestingly, spindle-shaped cells positive for vimentin were found in the CD44(high) /ALDH1(high) but not the CD44(high) /ALDH1(low) cell population. In addition, the ALDH1 activity and sphere forming ability of CD44(high) /ALDH1(high) cells was significantly inhibited by GSK3ß knockdown. On the other hand, CD44(high) /ALDH1(low) cells exhibited high epidermal growth factor receptor (EGFR) expression and increased cell growth. CONCLUSIONS: Our results show that GSK3ß plays a major role in maintenance of stemness of CD44(high) /ALDH1(high) HNSCC cells. Additionally, they indicate a close relationship between CSC and mesenchymal characteristics in HNSCC.

Clinicopathological analysis of salivary gland carcinomas and literature review.

Malignant salivary gland tumors are rare and exhibit a broad spectrum of phenotypic heterogeneity. The objective of this study was to investigate prognostic factors in patients with salivary gland carcinomas and review the results in light of other reports. We retrospectively reviewed 40 patients with primary salivary gland carcinomas who were diagnosed and treated at our institution between 1991 and 2014. Of the 40 tumors, 19 (47.5%) were mucoepidermoid carcinomas, 11 (27.5%) were adenoid cystic carcinomas, 7 (17.5%) were acinic cell carcinomas, 2 (5.0%) were myoepithelial carcinomas and 1 (2.5%) was a squamous cell carcinoma. Clinically positive lymph nodes were present in 4 patients (10.0%). As regards clinical stage, 15 cases (37.5%) were stage I, 13 (32.5%) were stage II, 1 (2.5%) was stage III and 11 (27.5%) were stage IVA. The majority of the patients (97.5%) were treated with surgery, of whom 25 (62.5%) received surgery alone and 14 (35.0%) underwent surgery in combination with chemotherapy or chemotherapy and radiotherapy. The median follow-up time for all the patients was 48 months. The disease-specific survival rate at 5 years was 87.1%. We identified a significant correlation between poor survival rate and histological grade (intermediate/high), tumor size (T3/T4), lymph node metastasis (node-positive) and clinical stage (III/IV) using the Kaplan-Meier method (P<0.05 for each). In addition, the Cox proportional hazards regression analysis confirmed that lymph node metastasis and tumor size were independent prognostic factors for disease-specific survival (hazard ratio = 18.7 and 15.1, respectively; P=0.023 and 0.037, respectively). Furthermore, tumor size was found to be a predictive factor regarding recurrence in the multivariate logistic regression analysis (odds ratio = 8.35; P=0.025). Our results suggest that lymph node metastasis and tumor size are significant prognostic factors for patients with salivary gland carcinomas.