Mechanical forces induce odontoblastic differentiation of mesenchymal stem cells on three-dimensional biomimetic scaffolds.

The mechanical induction of cell differentiation is well known. However, the effect of mechanical compression on odontoblastic differentiation remains to be elucidated. Thus, we first determined the optimal conditions for the induction of human dental pulp stem cells (hDPSCs) into odontoblastic differentiation in response to mechanical compression of three-dimensional (3D) scaffolds with dentinal tubule-like pores. The odontoblastic differentiation was evaluated by gene expression and confocal laser microscopy. The optimal conditions, which were: cell density, 4.0 × 105 cells/cm2 ; compression magnitude, 19.6 kPa; and loading time, 9 h, significantly increased expression of the odontoblast-specific markers dentine sialophosphoprotein (DSPP) and enamelysin and enhanced the elongation of cellular processes into the pores of the membrane, a typical morphological feature of odontoblasts. In addition, upregulation of bone morphogenetic protein 7 (BMP7) and wingless-type MMTV integration site family member 10a (Wnt10a) was observed. Moreover, the phosphorylation levels of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 were also enhanced by mechanical compression, indicating the involvement of the MAPK signalling pathway. It is noteworthy that human mesenchymal stem cells (MSCs) derived from bone marrow and amnion also differentiated into odontoblasts in response to the optimal mechanical compression, demonstrating the importance of the physical structure of the scaffold in odontoblastic differentiation. Thus, odontoblastic differentiation of hDPSCs is promoted by optimal mechanical compression through the MAPK signalling pathway and expression of the BMP7 and Wnt10a genes. The 3D biomimetic scaffolds with dentinal tubule-like pores were critical for the odontoblastic differentiation of MSCs induced by mechanical compression. Copyright © 2014 John Wiley & Sons, Ltd.

Trophic Effects of Dental Pulp Stem Cells on Schwann Cells in Peripheral Nerve Regeneration.

Recently, mesenchymal stem cells have demonstrated a potential for neurotrophy and neurodifferentiation. We have recently isolated mobilized dental pulp stem cells (MDPSCs) using granulocyte-colony stimulating factor (G-CSF) gradient, which has high neurotrophic/angiogenic potential. The aim of this study is to investigate the effects of MDPSC transplantation on peripheral nerve regeneration. Effects of MDPSC transplantation were examined in a rat sciatic nerve defect model and compared with autografts and control conduits containing collagen scaffold. Effects of conditioned medium of MDPSCs were also evaluated in vitro. Transplantation of MDPSCs in the defect demonstrated regeneration of myelinated fibers, whose axons were significantly higher in density compared with those in autografts and control conduits only. Enhanced revascularization was also observed in the MDPSC transplants. The MDPSCs did not directly differentiate into Schwann cell phenotype; localization of these cells near Schwann cells induced several neurotrophic factors. Immunofluorescence labeling demonstrated reduced apoptosis and increased proliferation in resident Schwann cells in the MDPSC transplant compared with control conduits. These trophic effects of MDPSCs on proliferation, migration, and antiapoptosis in Schwann cells were further elucidated in vitro. The results demonstrate that MDPSCs promote axon regeneration through trophic functions, acting on Schwann cells, and promoting angiogenesis.

Isolation of a stable subpopulation of mobilized dental pulp stem cells (MDPSCs) with high proliferation, migration, and regeneration potential is independent of age.

Insights into the understanding of the influence of the age of MSCs on their cellular responses and regenerative potential are critical for stem cell therapy in the clinic. We have isolated dental pulp stem cells (DPSCs) subsets based on their migratory response to granulocyte-colony stimulating factor (G-CSF) (MDPSCs) from young and aged donors. The aged MDPSCs were efficiently enriched in stem cells, expressing high levels of trophic factors with high proliferation, migration and anti-apoptotic effects compared to young MDPSCs. In contrast, significant differences in those properties were detected between aged and young colony-derived DPSCs. Unlike DPSCs, MDPSCs showed a small age-dependent increase in senescence-associated ß-galactosidase (SA-ß-gal) production and senescence markers including p16, p21, Interleukin (IL)-1ß, -6, -8, and Groα in long-term culture. There was no difference between aged and young MDPSCs in telomerase activity. The regenerative potential of aged MDPSCs was similar to that of young MDPSCs in an ischemic hindlimb model and an ectopic tooth root model. These results demonstrated that the stem cell properties and the high regenerative potential of MDPSCs are independent of age, demonstrating an immense utility for clinical applications by autologous cell transplantation in dental pulp regeneration and ischemic diseases.

The use of granulocyte-colony stimulating factor induced mobilization for isolation of dental pulp stem cells with high regenerative potential.

Human dental pulp stem cells (DPSCs) contain subsets of progenitor/stem cells with high angiogenic, neurogenic and regenerative potential useful for cell therapy. It is essential to develop a safe and efficacious method to isolate the clinical-grade DPSCs subsets from a small amount of pulp tissue without using conventional flow cytometry. Thus, a method for isolation of DPSCs subsets based on their migratory response to optimized concentration of 100 ng/ml of granulocyte-colony stimulating factor (G-CSF) was determined in this study. The DPSCs mobilized by G-CSF (MDPSCs) were enriched for CD105, C-X-C chemokine receptor type 4 (CXCR-4) and G-CSF receptor (G-CSFR) positive cells, demonstrating stem cell properties including high proliferation rate and stability. The absence of abnormalities/aberrations in karyotype and lack of tumor formation after transplantation in an immunodeficient mouse were demonstrated. The conditioned medium of MDPSCs exhibited anti-apoptotic activity, enhanced migration and immunomodulatory properties. Furthermore, transplantation of MDPSCs accelerated vasculogenesis in an ischemic hindlimb model and augmented regenerated pulp tissue in an ectopic tooth root model compared to that of colony-derived DPSCs, indicating higher regenerative potential of MDPSCs. In conclusion, this isolation method for DPSCs subsets is safe and efficacious, having utility for potential clinical applications to autologous cell transplantation.

Polymorphisms of apolipoprotein e and methylenetetrahydrofolate reductase in the Japanese population.

AIM: The aim of this study is to analyze the effect of apolipoprotein E (apo E) and methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms on serum lipid and homocysteine levels in the general Japanese population. METHODS: We analyzed the polymorphisms in individuals randomly selected from among participants of Serum Lipid Survey 2000. RESULTS: The frequency of the epsilon2, epsilon3, and epsilon4 alleles of APOE was 4.2, 85.3, and 10.5%, respectively. Individuals with the genotype epsilon4/epsilon4 had the highest total and low-density lipoprotein (LDL) cholesterol levels, while those with epsilon2/epsilon2 had the lowest. Individuals with the epsilon2/epsilon2 and epsilon2/epsilon4 genotypes had higher remnant-like particles (RLP)-cholesterol levels than those with epsilon2epsilon3, epsilon3epsilon3, and epsilon3epsilon4. There was a trend for individuals with the epsilon2/epsilon4 and epsilon2/epsilon2 genotypes to have higher triglyceride levels, although the difference was not significant. The presence of the T allele in a MTHFR polymorphism (C667T) was associated with higher homocysteine levels, which is more prominent in men than in women. CONCLUSION: Thus in our large-scale analysis we have shown that RLP-cholesterol is better associated with, APOE genotype than triglyceride and the effect of the T allele on MTHFR polymorphism (C667T) homocysteine levels is more prominent in men than in women among Japanese.

Prevalence of metabolic syndrome in the general Japanese population in 2000.

To determine the prevalence of metabolic syndrome in the Japanese general population, we analyzed data from a nationwide survey conducted in 2000. According to the Japanese new diagnostic criteria for metabolic syndrome in 2005, we analyzed 3,264 people aged from 20 to 79 (men, 1,917; women, 1,347) from the total participants. The incidence of metabolic syndrome was 7.8%. Men had a higher incidence (12.1%) than women (1.7%). Most of the women satisfying the criteria were 50 years old or over, while the incidence in men started to rise from their 30s. When we applied the criteria of Adult Treatment Panel III, the incidence was about 3-fold higher. In this population visceral obesity was associated with metabolic abnormalities, such as higher LDL-cholesterol, triglyceride, glucose, and blood pressure and lower HDL-cholesterol. Thus we determined the incidence of metabolic syndrome and each metabolic abnormality in the Japanese general population in 2000 and found an association of visceral obesity with metabolic abnormalities. Intervention to reduce the incidence of metabolic syndrome in Japan is necessary to reduce the risk of cardiovascular disease.

Polymorphisms in four genes related to triglyceride and HDL-cholesterol levels in the general Japanese population in 2000.

We studied the association of six common polymorphisms of four genes related to lipid metabolism with serum lipid levels. We selected single-nucleotide polymorphisms (SNPs) in the genes for cholesteryl ester transfer protein (CETP), lipoprotein lipase (LPL), hepatic lipase (LIPC), and apolipoprotein CIII (APOC3), and studied 2267 individuals randomly selected from the participants of Serum Lipid Survey 2000. There was a significant association of CETP polymorphism (D442G, Int14 +1 G --> A, and TaqIB), LPL polymorphism (S447X), and LIPC polymorphism (-514 --> CT) with HDL-cholesterol levels. We also found a significant association of LPL polymorphism (S447X) and APOC3 polymorphism (SstI) with triglyceride levels. This is the largest database showing the association of common genetic variants in lipid metabolism with serum lipid levels in the general Japanese population. Further study is necessary to elucidate the role of these gene polymorphisms in cardiovascular events.

A nineteen-year cohort study on the relationship of electrocardiographic findings to all cause mortality among subjects in the national survey on circulatory disorders, NIPPON DATA80.

BACKGROUND: Electrocardiogram (ECG) is one of the most popular tools for daily clinics and health checkup, and has been used for the National Survey on Circulatory Disorders to assess the health status in Japanese people. The meaningfulness to predict mortality from all causes among people with ECG abnormality is to be clarified using national samples. METHODS: ECG findings recorded among 9,638 subjects for National Survey on Circulatory Disorders 1980 in Japan, were classified using the Minnesota Codes (mc). Their relationships to all cause mortality over 19 years were examined using Cox proportional hazard models adjusting for sex, age, systolic blood pressure, blood glucose, and smoking habits. RESULTS: Subjects with abnormal Q-QS findings showed significantly high hazard ratios ( 3.71(mc1-1) and 1.57 (mc1-3)) for mortality to the subjects who were free from any major ECG findings. Hazard ratios were 1.37 (mc2-1) to 4.16 (mc2-5) for axis deviation, and 1.34 (mc3-1) to 1.35 (mc3-3) for left high R waves. Those were 1.63 (mc4-3) to 2.59 (mc4-1) for ST depression, and 1.54 (mc5-3) to 2.33 (mc5-1) for T abnormality. The lower the second number of the Minnesota Codes was, the higher hazard ratio was observed in the Q-QS, ST, T codes. The hazard ratios of junction-type ST depression (mc4-4), and low T waves (mc5-4, 5-5) were not significant. CONCLUSIONS: ECG findings defined by the Minnesota Codes were useful to predict the risk for mortality from all causes even after adjusting for the other major risk factors, and the results supported a usefulness of the ECG for health check-ups.

Serum lipid survey and its recent trend in the general Japanese population in 2000.

To determine the recent serum lipid levels and other serum variables in the general Japanese population and trends in their changes over the past 40 years, a nationwide survey of serum lipid levels was conducted in 36 institutes from various districts around Japan in 2000. The total number of subjects was 12,839, aged 4 through 99 years. The mean total cholesterol level was 201 mg/dl; 202 mg/dl in men and 200 mg/dl in women. The mean HDL-cholesterol level was 59 mg/dl; 55 mg/dl in men and 65 mg/dl in women. The mean LDL-cholesterol level was 118 mg/dl; 121 mg/dl in men and 115 mg/dl in women. The mean triglyceride level was 118 mg/dl; 136 mg/dl in men and 92 mg/dl in women. The total cholesterol level slightly increased by 5 mg/dl in 10 years. Although the triglyceride level in women did not change, the triglyceride level in men increased over 10 years, especially in the 30s through 70s age bracket, indicating a possible increase in metabolic syndromes in the future. The present results will become the standard serum lipid level data for the Japanese people, and succeeding 10-year surveys will clarify the trends of lipid levels in this country.

Life style and cardiovascular risk factors in the Japanese population--from an epidemiological survey on serum lipid levels in Japan 1990 part 1: influence of life style and excess body weight on HDL-cholesterol and other lipid parameters in men.

Low HDL-cholesterol (HDL-C) has long been used as an important predictor of coronary artery disease (CAD), although HDL-C values themselves are influenced by various factors including serum triglyceride (TG) levels, obesity, and life style. In view of the importance of the metabolic syndrome as a risk factor of CAD, changes in HDL-C and other lipid parameters in the Japanese population associated with life style, especially in males, were analyzed in this study based on data obtained in an epidemiological survey carried out in 1990. Smokers had higher TG and lower HDL-C levels than non-smokers, while BMI and LDL-C were slightly decreased by smoking in middle-aged men (40-59 years old). Increases in both HDL-C and TG due to alcohol consumption were associated with an increase in BMI in younger men aged 20-39. In middle-aged men, significant increases in HDL-C were seen in every quintile of BMI, while the increase in TG levels due to alcohol was small. Middle-aged men engaged in occupations requiring greater physical activity also had higher HDL-C levels in every quintile of BMI. The influence of life style on serum lipid parameters appeared to be mostly expressed as a function of BMI in younger men, while it appeared to be independent of BMI in older men.

Life style and cardiovascular risk factors in the Japanese population--from an epidemiological survey on serum lipid levels in Japan 1990 part 2: association of lipid parameters with hypertension.

The importance of metabolic syndrome as a risk factor of coronary artery disease (CAD) has recently become more and more recognized. In view of such a background, changes in cholesterol and other lipid parameters in the Japanese population associated with excess body weight and hypertension were analyzed in this study based on data obtained in an epidemiological survey carried out in 1990. Hypertension was closely associated with a higher body mass index (BMI). Among the lipid parameters, triglyceride (TG) and non-HDL-C (total cholesterol minus HDL-cholesterol) levels showed remarkable differences between hypertensive and normotensive subjects, with a greater statistical significance than LDL-cholesterol (LDL-C) levels. Changes in lipid parameters in the presence of hypertension were mostly associated with an increase in BMI in younger men aged 20-39, while increases in TG levels took place independently of excess body weight in middle-aged (40-59-year old) men. Considering that hypertension is a common and the greatest risk factor in the Japanese population, TG and non-HDL-C appear to be more important than LDL-C as major lipid parameters related to atherogenesis.

Impact of elevated blood pressure on mortality from all causes, cardiovascular diseases, heart disease and stroke among Japanese: 14 year follow-up of randomly selected population from Japanese -- Nippon data 80.

The objectives of the study were to clarify the relationship between blood pressure and mortality from stroke, heart disease, cardiovascular diseases and all causes of death among representative population of Japanese and to estimate category-specific excess mortality from stroke due to blood pressure (BP) level. The study design comprised a retrospective cohort study using the 1980 National Survey on Cardiovascular Diseases and identification of underlying causes of death using national vital statistics data. In 1994, a 14-year follow-up cohort study was conducted among participants of the National Survey on Cardiovascular Diseases in 1980, randomly selected from the Japanese population. With a collaboration of 300 public health centres, which had conducted the original survey in 1980, 91.4% of the participants of the original survey could be followed up. Total observed person-years were 53948 for men and 70932 for women. During follow-up, 1327 deaths were observed. BP levels were significantly related to mortality from strokes, cardiovascular diseases and all causes of death for both sexes (P<0.001). Heart disease mortality was significantly related to BP levels among men (P<0.05) while not among women. Estimated excess mortality was 130% for men and 42% for women and chiefly observed among moderate hypertensives (48% for men and 16% for women). In conclusion, high blood pressure was a risk factor for mortality from all causes as well as those from cardiovascular diseases, stroke and heart disease among Japanese. Since the major part of excess mortality was due to mild hypertension, a population strategy to reduce blood pressure should be encouraged.

Risk factors for coronary heart disease in the Japanese--comparison of the background of patients with acute coronary syndrome in the ASPAC study with data obtained from the general population. Asia-Pacific Collaboration on CHD Risk Factor Intervention study.

The relative importance of metabolic risk factors for coronary heart disease (CHD) in the Japanese is assessed by comparing their prevalence in patients with acute coronary syndrome (ACS) enrolled in the Asia-Pacific Collaboration on CHD Risk Factor Intervention (ASPAC) study to that obtained by a serum lipid survey carried out in 1990 and also by comparing them to the ASPAC data from other countries and regions in this area. Hypertension was the most prevalent risk factor among Japanese patients with ACS as in the other countries and regions. The prevalence of obesity with a body mass index (BMI) of 30 or more was several times higher than that in the general population, although the rate was still much lower than in New Zealand and Singapore. In addition to hypercholesterolemia, hypertriglyceridemia and diabetes mellitus were frequently found in Japanese patients with ACS. When the prevalence of metabolic risk factors was compared between people with and without hypertension in the general population, the most remarkable difference was seen in BMI, followed by triglyceride and total cholesterol. These results indicate that hypertriglyceridemia and diabetes mellitus may be more important CHD risk factors in the Japanese population than LDL-cholesterol.