Spheroid culture enhances osteogenic potential of periodontal ligament mesenchymal stem cells.

OBJECTIVE AND BACKGROUND: Human periodontal ligament mesenchymal stem cells (hPDLMSCs) are reported to be responsible for homeostasis and regeneration of periodontal tissue. Although hPDLMSCs are commonly cultured in monolayers, monolayer cultures have been reported as inferior to 3-dimensional cultures such as spheroids, which are spherical clusters of cells formed by self-assembly. The aim of this study was to examine the osteogenic phenotype of spheroids of hPDLMSCs, compared with monolayer cultures of hPDLMSC, in vitro and in vivo. MATERIAL AND METHODS: Spheroids were formed using microwell chips that were tagged with polyethylene glycol. Mesenchymal stem cell (MSC) markers in hPDLMSC spheroids were examined by flow cytometer. Real-time polymerase chain reaction analysis was examined to measure the expressions of stemness markers and osteogenesis-related genes in monolayer and spheroid-cultured hPDLMSCs. Immunofluorescence analysis was performed to confirm protein expressions of stemness markers in PDLMSC spheroids. Nodule formation assay, alkaline phosphatase (ALP) activity assay and transplantation assay in a mouse calvarial defect model were performed to confirm the osteogenic potential of hPDLMSC spheroids. To elucidate the mechanism of spheroid culture enhanced osteogenesis in hPDLMSCs with osteoinductive medium (OIM), a small interfering RNA (siRNA) assay targeted with secreted frizzled-related protein 3 (SFRP3) was examined. The levels of SFRP3 expression in monolayer and spheroid-cultured hPDLMSCs with OIM were measured by real-time polymerase chain reaction and western blotting analysis. ALP gene expression and ALP activity were examined in SFRP3-deficient hPDLMSC spheroids. RESULTS: The hPDLMSC spheroids expressed MSC markers, which were similar to hPDLMSCs grown in monolayer cultures. Intriguingly, the protein and mRNA expressions of transcription factors that regulate "stemness" were significantly increased in hPDLMSC spheroids, compared with hPDLMSCs in monolayer cultures. Nodule formation by hPDLMSCs was significantly increased in spheroid cultures grown with OIM, compared with monolayer-cultured hPDLMSCs. ALP activity and expression of osteogenesis-related genes were also significantly enhanced in hPDLMSC spheroids, compared with monolayer cultures. Treatment with hPDLMSC spheroids significantly enhanced new bone formation in a murine calvarial defect model, compared with hPDLMSCs in monolayer culture. Finally, to elucidate mechanisms by which spheroid culture enhances ALP activation in hPDLMSCs grown with OIM, an siRNA assay was used to manipulate expression of SFRP3, a Wnt signaling antagonist. Knockdown of SFRP3 suppressed ALP gene expression in hPDLMSCs grown in OIM; further, it suppressed ALP activity in spheroid culture. These data suggest that the enhancement of osteogenic potential in hPDLMSC spheroids is regulated through SFRP3-mediated ALP activation. CONCLUSION: Spheroid cultures of hPDLMSCs may be a novel and useful tool in regenerative medicine.

Associations among tooth loss, systemic inflammation and antibody titers to periodontal pathogens in Japanese patients with cardiovascular disease.

BACKGROUND AND OBJECTIVE: It is well known that there is a strong relationship between periodontitis and cardiovascular disease (CVD). Tooth loss reflects an end-stage condition of oral diseases, such as periodontitis. Infection with specific periodontal pathogens is known as a possible factor that influences development of CVD. The aim of this study was to assess the relationship between the number of residual teeth and systemic inflammatory conditions in patients with CVD. MATERIAL AND METHODS: We divided 364 patients with CVD into four groups, according to the number of residual teeth: (i) ≥20 teeth; (ii) 10-19 teeth; (iii) 1-9 teeth; and (iv) edentulous. We recorded medical history, blood data and periodontal conditions. Serum samples were obtained and their IgG titers against three major periodontal pathogens were measured. RESULTS: Smoking rate and the prevalence of diabetes mellitus were higher in edentulous patients and in subjects with a few teeth compared with patients with many teeth. The levels of C-reactive protein were higher in patients with 1-9 teeth than in those with 10-19 teeth and with ≥20 teeth. The level of Porphyromonas gingivalis IgG in the group with 10-19 teeth was statistically higher than that in the group with ≥20 teeth. The level of P. gingivalis IgG in the edentulous group tended to be lower than that in the other groups. CONCLUSION: The patients with 1-9 teeth had the highest level of C-reactive protein among the four groups, and the patients with 10-19 teeth had the highest level of IgG to periodontal bacteria. We conclude that the number of remaining teeth may be used to estimate the severity of systemic inflammation in patients with CVD.

Gingival epithelial cells support osteoclastogenesis by producing receptor activator of nuclear factor kappa B ligand via protein kinase A signaling.

BACKGROUND AND OBJECTIVE: Periodontal disease is dental plaque-induced inflammatory disease of the periodontal tissues that results in bone loss in the affected teeth. During bone resorption, receptor activator of nuclear factor kappa B ligand (RANKL) is an essential factor that regulates osteoclastogenesis. Recently, we found that gingival epithelial cells (GECs) in periodontal tissue produce RANKL, the expression of which is regulated by tumor necrosis factor-α and protein kinase A signaling. In this study, we asked whether RANKL-producing GECs induce bone marrow macrophages (BMMs) to form osteoclasts in a co-culture system. MATERIAL AND METHODS: Ca9-22 GECs and osteoclast precursor BMMs were co-cultured with or without the protein kinase A signaling activator forskolin or inhibitor H89 to examine whether the RANKL-producing GECs could be induced to form osteoclasts, as determined using a pit formation assay. RESULTS: Osteoclasts formed spontaneously in co-cultures of Ca9-22 cells and BMMs, even in the absence of RANKL. The cells were cultured on bone slices for 14 d, at which time resorption pits were observed. Forskolin treatment significantly increased osteoclast numbers in these co-cultures, but forskolin alone did not induce osteoclast formation by BMMs. CONCLUSION: GECs producing RANKL are able to support osteoclastogenesis in an in vitro co-culture system using GECs and BMMs, in a process promoted by forskolin.

Evaluation of two Japanese regulatory actions using medical information databases: a 'Dear Doctor' letter to restrict oseltamivir use in teenagers, and label change caution against co-administration of omeprazole with clopidogrel.

WHAT IS KNOWN AND OBJECTIVE: The implementation of appropriate epidemiological methodology using medical information databases (MIDs) to evaluate the effects of regulatory actions has been highly anticipated. To assess scientific methods for active pharmacovigilance using MIDs, we conducted a quantitative assessment of the impact of two regulatory actions by the Japanese government: (i) restriction of use of oseltamivir in teenagers in March 2007 and (ii) caution against the co-administration of omeprazole (OPZ) with clopidogrel (CPG) in April 2010. METHODS: Data were obtained from four hub hospitals in Japan. We measured the seasonal proportion of patients prescribed oseltamivir to those prescribed neuraminidase inhibitors for the 2002/2003 to 2010/2011 seasons. The monthly proportion of patients co-administered OPZ and CPG (OPZ+CPG) to those prescribed CPG was measured from May 2009 to April 2011. We evaluated the changes observed with implementation of the regulatory actions. To estimate the impact of the actions, we conducted segmented regression analysis using interrupted time series data. The impact was assessed by two parameter estimates of the regression model: the change in level for short-term effects and change in trend for long-term effects. RESULTS AND DISCUSSION: The use of oseltamivir in the target 10-19 years age group showed a significant and large decline (63·16%) immediately after the intervention (P = 0·0008). No change was observed in OPZ+CPG, although there was a relative inhibitory trend for OPZ+CPG compared with co-administration of lansoprazole or rabeprazole with CPG as the control group. When restricted to new users of CPG, the stratified results were consistent with the overall results. WHAT IS NEW AND CONCLUSION: The current analysis demonstrates the effectiveness of two regulatory actions. The results of the current study indicate that MID research can contribute to assessing and improving pharmacovigilance activities.

An algorithm for the identification of heparin-induced thrombocytopenia using a medical information database.

WHAT IS KNOWN AND OBJECTIVE: Using effective algorithms for extracting relevant drug and patient information from electronic medical information data systems is likely to form an increasingly important aspect of pharmacovigilance. To this end, we aimed to develop and validate a novel algorithm for detecting heparin-induced thrombocytopenia (HIT) using a medical information database (MID) and for identifying possible risk factors for HIT. METHODS: We developed a new algorithm for detecting HIT based on platelet count at distinct time-points and diagnostic information from patients treated with unfractionated heparin (UFH) from April 2008 through March 2012 at Hospital of Hamamatsu University School of Medicine, Japan. Definitive diagnoses of HIT were made through reviews of the medical records by a skilled haematologist. The performance of the algorithm was assessed using the positive predictive value (PPV). Multivariate logistic regression analysis was used to identify possible risk factors for HIT. RESULTS AND DISCUSSION: This algorithm detected 47 patients with suspected HIT in the source population (n = 2875). Of these, 41 were identified as patients with definitive HIT after review of the medical records. The PPV for the algorithm was 87·2%, and the frequency of definitive HIT was 1·4%. Longer-term treatment (≥4 days) with UFH was identified as a risk factor for HIT, with an odds ratio of 5·38 (95% CI: 2·35-12·32) for definitive HIT. WHAT IS NEW AND CONCLUSION: We developed a novel, high-PPV detection algorithm for HIT and identified longer-term treatment with UFH as a risk factor for HIT. Our results support the utility of MIDs for improving pharmacovigilance.

Key factors for the dynamic ETEM observation of single atoms.

We present further modifications to aberration-corrected environmental transmission electron microscopy (AC-ETEM) for the dynamic HRTEM observation of single atoms. Additional pumping levels that include three additional turbomolecular pumps (TMPs) enable a base pressure of 3.5 × 10(-5) Pa in the sample chamber. The effect of these additional TMPs on image resolution was measured in reciprocal space using information limit (Young's fringes) on a standard cross grating sample and also with platinum (Pt) single atoms on an amorphous carbon film (Pt/a-carbon). The Pt/a-carbon was used for measuring the effect of gas pressure on single-atom imaging in addition to the evaluation of vibrations of TMPs, samples, magnetic lenses and a microscope column of the AC-ETEM. TMPs did not affect the ETEM imaging performance when an anti-vibration table was used, and 0.10-nm resolution was achieved. Dynamic ETEM observation of Pt single atoms was achieved in 4.0 × 10(-2) Pa of air, using a modified AC-ETEM system and a high-speed CCD camera with a time resolution of 0.05 s.

Development of a detection algorithm for statin-induced myopathy using electronic medical records.

WHAT IS KNOWN AND OBJECTIVE: Demonstration of the utility of electronic medical records (EMRs) for pharmacovigilance (PV) has been highly anticipated. Analysis using appropriately selected EMRs should enable accurate estimation of adverse drug event (ADE) frequencies and thus promote appropriate regulatory actions. Statin-induced myopathy (SIM) is a clinically important ADE, but pharmacoepidemiological methodology for detecting this ADE with high predictability has not yet been established. This study aimed to develop a detection algorithm, highly selective for SIM using EMRs. METHODS: We collected EMRs on prescriptions, laboratory tests, diagnoses and medical practices from the hospital information system of Kobe University Hospital, Japan, for a total of 5109 patients who received a statin prescription from April 2006 to March 2009. The current algorithm for extracting SIM-suspected patients consisted of three steps: (i) event detection: increase in creatine kinase (CK) and subsequent statin discontinuation, (ii) filtration by exclusion factors (disease diagnosis/medical practices) and (iii) refinement by the time course of CK values (baseline, event and recovery). A causal relationship between the event and statin prescription (probable/possible/unlikely) was judged by review of patient medical charts by experienced pharmacists. The utility of the current algorithm was assessed by calculating the positive predictive value (PPV). In a comparative analysis, subjects screened in step 1 were extracted by the diagnostic term/code for 'myopathy/rhabdomyolysis', and the PPV of this diagnostic data approach was also estimated. RESULTS AND DISCUSSION: Five subjects with suspected SIM were identified using our proposed algorithm, giving a frequency of 0·1% for the adverse event. Review of the medical charts revealed that the causal association of SIM with statin use was judged as 'Likely (probable/possible)' for all five suspected patients; thus, the PPV was estimated as 100% (95% confidence interval: 56·6-100%). The higher utility of the current algorithm compared with the diagnostic data approach was also shown by assessing the PPV (100 vs. 33·3%). WHAT IS NEW AND CONCLUSION: We report on a detection algorithm with high predictability for SIM using EMRs. Combined use of exclusion criteria for disease, medical practice data and time course of CK values contributes to better prediction of SIM. The utility of the proposed algorithm should be further confirmed in a larger study.

Clarithromycin suppresses the periodontal bacteria-accelerated abdominal aortic aneurysms in mice.

BACKGROUND AND OBJECTIVE: Although clarithromycin (CAM) has many biological functions, including regulation of MMPs, little is known about its effect on abdominal aortic aneurysms. Periodontopathic bacteria have been reported to be associated with several kinds of circulatory diseases. The purpose of this study was therefore to clarify the effect of CAM on periodontopathic bacteria-accelerated abdominal aortic aneurysms. MATERIAL AND METHODS: Abdominal aortic aneurysm was produced in mice by the peri-aortic application of 0.25 m CaCl(2). The mice were inoculated once per week with live Porphyromonas gingivalis, which is one of the major periodontopathic bacteria. Test mice (n=8) were given a daily oral dose of CAM, while control mice (n=13) were not. RESULTS: Four weeks after the operation, the P. gingivalis-injected and CAM-treated mice showed a significant decrease in the aortic diameter in comparison with the mice only injected with P. gingivalis. Histopathologically, the samples obtained from the P. gingivalis-injected and CAM-treated mice showed less elastic degradation. Moreover, the plasma MMP-2 concentration of the CAM-treated mice decreased significantly. CONCLUSION: These findings suggest that CAM administration is useful to suppress periodontal bacteria-accelerated abdominal aortic aneurysms via MMP regulation.

Porphyromonas gingivalis promotes murine abdominal aortic aneurysms via matrix metalloproteinase-2 induction.

BACKGROUND AND OBJECTIVE: Abdominal aortic aneurysm (AAA) is a common and lethal disorder, and MMPs are highly expressed in AAA lesions. Large numbers of periodontopathic bacteria have been reported to be present in specimens obtained from the aortic walls of patients with an AAA. The purpose of this study was to analyze the influence of periodontopathic bacteria on AAA dilatation. MATERIAL AND METHODS: AAAs were produced in mice by the periaortic application of 0.25 M CaCl(2), and NaCl was used as a control. The mice were inoculated once weekly with live Porphyromonas gingivalis, live Aggregatibacter actinomycetemcomitans or vehicle. RESULTS: Four weeks after the periaortic application of either CaCl(2) or NaCl, a significant increase was observed in the aortic diameter of P. gingivalis-challenged mice compared with the vehicle control mice (p < 0.05), whereas there was no statistically significant increase in the aortic diameter of the A. actinomycetemcomitans-challenged mice. Immunohistochemical analysis found significantly higher numbers of CD8-positive and MOMA2-positive cells and significantly higher levels of MMP-2 in the aneurysmal samples of P. gingivalis-challenged mice compared with control mice. Live P. gingivalis promoted a significant proliferation of splenocytes in comparison with P. gingivalis-lipopolysaccharide and live A. actinomycetemcomitans (p < 0.05). CONCLUSION: These findings demonstrate that challenge with P. gingivalis, but not with A. actinomycetemcomitans, can accelerate, or even initiate, the progression of experimental AAA through the increased expression of MMPs.

CYP2C9*3 influences the metabolism and the drug-interaction of candesartan in vitro.

Candesartan cilexetil is an angiotensin II receptor antagonist, and candesartan, its active metabolite, is metabolized by CYP2C9. However, the effect of CYP2C9*3 on candesartan metabolism is not established. We characterized the kinetics of candesartan by CYP2C9*1/*1 and CYP2C9*1/*3 in human liver microsomes. The difference between the two was not significant. Subsequently, CYP2C9*1 and CYP2C9*3 (Leu359) were expressed in yeast, and the kinetics of candesartan were determined. The wild-type showed the lower Km (345 vs 439 microM; 3/4) and higher Vmax/Km (1/3) than the Leu359 variant. Also, we investigated potential interaction between candesartan and warfarin with both the wild-type and the Leu359 variant. Candesartan had no effect on S-warfarin 7-hydroxylation. In contrast, S-warfarin inhibited candesartan metabolism by the wild-type (K = 17microM) greater than by the Leu359 variant (Ki = 36 microM). These findings suggest that CYP2C9*3 may change not only the metabolic activity but also the inhibitory susceptibility compared with CYP2C9*1.