Exosomes derived from dental pulp stem cells accelerate cutaneous wound healing by enhancing angiogenesis via the Cdc42/p38 MAPK pathway.

Skin wound healing is a common challenging clinical issue which requires advanced treatment strategies. The present study investigated the therapeutic effects of exosomes derived from dental pulp stem cells (DPSC­Exos) on cutaneous wound healing and the underlying mechanisms. The effects of DPSC­Exos on cutaneous wound healing in mice were examined by measuring wound closure rates, and using histological and immunohistochemical analysis. A series of functional assays were performed to evaluate the effects of DPSC­Exos on the angiogenic activities of human umbilical vein endothelial cells (HUVECs) in vitro. Tandem mass tag­based quantitative proteomics analysis of DPSCs and DPSC­Exos was performed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were used to evaluate the biological functions and pathways for the differentially expressed proteins in DPSC­Exos. Western blot analysis was used to assess the protein levels of cell division control protein 42 (Cdc42) and p38 in DPSC­Exos and in HUVECs subjected to DPSC­Exos­induced angiogenesis. SB203580, a p38 mitogen­activated protein kinase (MAPK) signaling pathway inhibitor, was employed to verify the role of the p38 MAPK pathway in vitro and in vivo. Histological and immunohistochemical staining revealed that the DPSC­Exos accelerated wound healing by promoting neovascularization. The DPSC­Exos promoted the migration, proliferation and capillary formation capacity of HUVECs. Proteomics data demonstrated that proteins contained in DPSC­Exos regulated vasculature development and angiogenesis. Pathway analysis revealed that proteins expressed in DPSC­Exos were involved in several pathways, including MAPK pathway. Western blot analysis demonstrated that the DPSC­Exos increased the protein levels of Cdc42 and phosphorylation of p38 in HUVECs. SB203580 suppressed the angiogenesis induced by DPSC­Exos. On the whole, the present study demonstrates that DPSC­Exos accelerate cutaneous wound healing by enhancing the angiogenic properties of HUVECs via the Cdc42/p38 MAPK signaling pathway.

MicroRNA-enriched small extracellular vesicles possess odonto-immunomodulatory properties for modulating the immune response of macrophages and promoting odontogenesis.

BACKGROUND: To investigate the odonto-immunomodulatory properties of dental pulp stem cell-derived small extracellular vesicles (DPSCs-sEV), which promote odontogenesis by switching macrophages toward the pro-healing M2 phenotype. METHODS: MicroRNA sequencing was carried out for microRNA profiling of DPSCs-sEV. Automated Western blot, qPCR, ELISA, and flow cytometry were performed to identify the functions of microRNA-enriched DPSCs-sEV in macrophages. A luciferase reporter gene assay was carried out to confirm exosomal miR-125a-3p's direct target gene. DPSCs-sEV-stimulated macrophage-conditioned media were used to promote odontogenesis in DPSCs and explore the mechanism of immune response in DPSCs-SEV-stimulated odontogenesis. DPSCs-sEV were injected into the exposed pulp tissue of rat incisor to investigate the odonto-immunomodulatory properties of DPSCs-sEV in vivo. RESULTS: DPSCs-sEV switched macrophages to the pro-healing M2 phenotype by inhibiting TLR and NFκΒ signaling. MicroRNA sequencing found 81 microRNAs significantly altered in DPSCS-sEV, with miR-125a-3p showing a 12-fold upregulation. Exosomal miR-125a-3p switched macrophages toward the M2 phenotype via inhibiting NFκΒ and TLR signaling via direct IKBKB targeting. Interestingly, DPSCs-sEV and the encapsulated miR-125a-3p enhanced BMP2 release in macrophages, promoting odontogenesis in DPSCs through BMP2 pathway activation. The rat study confirmed that DPSCs-sEV could be used as ideal biomimetic tools to enhance odontogenesis by switching macrophages toward pro-healing M2 cells. CONCLUSIONS: We firstly defined the odonto-immunomodulatory properties of microRNA-enriched DPSCs-sEV, which could be used as ideal biomimetic tools to enhance odontogenesis by switching macrophages toward the pro-healing M2 phenotype.

Potential risk of certain cancers among patients with Periodontitis: a supplementary meta-analysis of a large-scale population.

Background: Some studies have reported biological linkages between periodontitis and esophageal cancer, prostate cancer, kidney cancer, hematological malignancy, and melanoma of the skin. This meta-analysis aimed to assess the relationship between periodontitis and the aforementioned five cancers. Methods: Eligible studies on the association between periodontitis and the aforementioned five kinds of cancers were retrieved. The statistical analysis was conducted using Stata 12.0. Results: Ten articles (more than 100,000 samples for most cancers) were included. With statistical significance, participants with periodontitis might have enhanced risks of esophageal cancer (HR = 1.79, 95% CI: 1.15-2.79), prostate cancer (HR = 1.20, 95% CI: 1.09-1.31), hematological malignancy (HR = 1.19, 95% CI: 1.09-1.29), and melanoma of skin (HR = 1.21, 95% CI: 1.03-1.42), compared with those without periodontitis. However, the evidence regarding the correlation between periodontitis and the susceptibility to kidney cancer was lacking (HR=1.30, 95% CI: 0.96-1.76). Conclusions: The present meta-analysis revealed a potential link between periodontitis and esophageal cancer, prostate cancer, hematological malignancy, and melanoma of the skin. However, multi-center studies with large sample sizes and multivariable adjustments are still needed to support the conclusion.

Three-dimensional position of mandibular third molars and its association with distal caries in mandibular second molars: a cone beam computed tomographic study.

OBJECTIVE: To analyze the prevalence of distal caries in mandibular second molars (M2Ms) and its correlation with the three-dimensional position of mandibular third molars (M3Ms) by cone beam computed tomography (CBCT) images. MATERIALS AND METHODS: CBCT scans involving 421 M3Ms were assessed. The presence of distal caries of M2Ms, patient age and gender, impaction depths and mesial angulation of M3Ms, the cementoenamel junction (CEJ) distances and contact point localization, and the horizontal positions of M3Ms were assessed. Risk factors were identified by multivariate logistic regression analysis. RESULTS: The overall prevalence of distal caries in M2Ms was 31.6%. Mesial angulation (16°-75°) of M3Ms, contact point localization at or below the CEJ of M2Ms, and a buccal or lingual position of M3Ms relative to the molar line were identified as risk factors for the prevalence of distal caries in M2Ms (p < 0.05). Distal caries was more severe when the mesial angulation of M3Ms was 16°-75° (p < 0.05). CONCLUSIONS: Buccal or lingual position of M3Ms may represent a new risk factor for the distal caries in M2Ms. Mesial angulation (16°-75°) of M3Ms is a predictive parameter for both the presence and severity of distal caries in M2Ms. CLINICAL RELEVANCE: As the presence of distal caries in M2Ms is significantly associated with the three-dimensional position of M3Ms, watchful monitoring or prophylactic removal of M3Ms should be deliberated when M3Ms are mesially angulated (16°-75°), buccally or lingually positioned, and with the contact point localization at or below the CEJ of M2Ms.

Magnesium-enriched microenvironment promotes odontogenic differentiation in human dental pulp stem cells by activating ERK/BMP2/Smads signaling.

BACKGROUND: Magnesium (Mg2+)-enriched microenvironment promotes odontogenic differentiation in human dental pulp stem cells (DPSCs), but the regulatory mechanisms remain undefined. The aim of this work was to assess magnesium's function in the above process and to explore the associated signaling pathway. METHODS: DPSCs underwent culture in odontogenic medium with the addition of 0, 1, 5, or 10 mM MgCl2. Intracellular Mg2+ levels in DPSCs were evaluated flow cytometrically using Mag-Fluo-4-AM. Mg2+-entry was inhibited by TRPM7 inhibitor 2-aminoethoxydiphenyl borate (2-APB). RNA-Sequencing was carried out for assessing transcriptome alterations in DPSCs during odontogenic differentiation associated with high extracellular Mg2+. KEGG pathway analysis was performed to determine pathways related to the retrieved differentially expressed genes (DEGs). Immunoblot was performed for assessing magnesium's role and exploring ERK/BMP2/Smads signaling. RESULTS: Mg2+-enriched microenvironment promoted odontogenic differentiation in DPSCs via intracellular Mg2+ increase. Consistently, the positive effect of high extracellular Mg2+ on odontogenic differentiation in DPSCs was blocked by 2-APB, which reduced Mg2+ entry. RNA-sequencing identified 734 DEGs related to odontogenic differentiation in DPSCs in the presence of high extracellular Mg2+. These DEGs participated in many cascades such as MAPK and TGF-ß pathways. Consistently, ERK and BMP2/Smads pathways were activated in DPSCs treated with high extracellular Mg2+. In agreement, ERK signaling inhibition by U0126 blunted the effect of high extracellular Mg2+ on mineralization and odontogenic differentiation in DPSCs. Interestingly, BMP2, BMPR1, and phosphorylated Smad1/5/9 were significantly decreased by U0126, indicating that BMP2/Smads acted as downstream of ERK. CONCLUSIONS: Mg2+-enriched microenvironment promotes odontogenic differentiation in DPSCs by activating ERK/BMP2/Smads signaling via intracellular Mg2+ increase. This study revealed that Mg2+-enriched microenvironment could be used as a new strategy for dental pulp regeneration.

Lineage-specific exosomes promote the odontogenic differentiation of human dental pulp stem cells (DPSCs) through TGFß1/smads signaling pathway via transfer of microRNAs.

BACKGROUND: Exosomes derived from dental pulp stem cells (DPSCs) can be used as biomimetic tools to induce odontogenic differentiation of stem cells, but the regulatory mechanisms and functions of exosome-encapsulated microRNAs are still unknown. The present study aimed to clarify the role of microRNAs contained in the exosomes derived from human DPSCs and their potential signaling cascade in odontogenic differentiation. METHODS: Exosomes were isolated from human DPSCs cultured undergrowth and odontogenic differentiation conditions, named UN-Exo and OD-Exo, respectively. The microRNA sequencing was performed to explore the microRNA profile contained in UN-Exo and OD-Exo. Pathway analysis was taken to detect enriched pathways associated with the predicted target genes of microRNAs. The regulatory roles of a highly expressed microRNA in OD-Exo were investigated through its inhibition or overexpression (miRNA inhibitors and miRNA mimics). Automated western blot was used to identify the function of exosomal microRNA and the roles of TGFß1/smads pathway in odontogenic differentiation of DPSCs. A luciferase reporter gene assay was used to verify the direct target gene of exosomal miR-27a-5p. RESULTS: Endocytosis of OD-Exo triggered odontogenic differentiation of DPSCs by upregulating DSP, DMP-1, ALP, and RUNX2 proteins. MicroRNA sequencing showed that 28 microRNAs significantly changed in OD-Exo, of which 7 increased and 21 decreased. Pathway analysis showed genes targeted by differentially expressed microRNAs were involved in multiple signal transductions, including TGFß pathway. 16 genes targeted by 15 differentially expressed microRNAs were involved in TGFß signaling. Consistently, automated western blot found that OD-Exo activated TGFß1 pathway by upregulating TGFß1, TGFR1, p-Smad2/3, and Smad4 in DPSCs. Accordingly, once the TGFß1 signaling pathway was inhibited by SB525334, protein levels of p-Smad2/3, DSP, and DMP-1 were significantly decreased in DPSCs treated with OD-Exo. MiR-27a-5p was expressed 11 times higher in OD-Exo, while miR-27a-5p promoted odontogenic differentiation of DPSCs and significantly upregulated TGFß1, TGFR1, p-Smad2/3, and Smad4 by downregulating the inhibitory molecule LTBP1. CONCLUSIONS: The microRNA expression profiles of exosomes derived from DPSCs were identified. OD-Exo isolated under odontogenic conditions were better inducers of DPSC differentiation. Exosomal microRNAs promoted odontogenic differentiation via TGFß1/smads signaling pathway by downregulating LTBP1.

MagT1 regulated the odontogenic differentiation of BMMSCs induced byTGC-CM via ERK signaling pathway.

BACKGROUND: Bone marrow mesenchymal stem cells (BMMSCs) are suitable cell sources for dental pulp regeneration, but the mechanism of BMMSCs differentiation into odontogenic lineage remains unknown. The aim of the present study was to reveal the role of magnesium transporter protein 1 (MagT1) and MAPK pathways in the odontogenic differentiation of BMMSCs. METHODS: The RNA sequencing (RNA-seq) was performed to explore the altered transcriptome of BMMSCs undergoing odontogenic differentiation induced by tooth germ cell-condition medium (TGC-CM). Pathway analysis was conducted to explore enriched pathways of the differential expression signature. Automated western blot, real-time PCR, shRNA lentivirus, and flow cytometry were used to detect the function of MagTl and MAPK pathway in odontogenic differentiation of BMMSCs. RESULTS: RNA-seq identified 622 differentially expressed genes associated with odontogenic differentiation of BMMSCs induced by TGC-CM, some of which were responsible for MAPK pathway. Consistently, we verified that TGC-CM induced odontogenic differentiation of BMMSCs through activating ERK/MAPK pathway, and the inactivation of ERK/MAPK pathway inhibited the odontogenic differentiation induced by TGC-CM. We also found MagT1 protein was significantly increased during odontogenic differentiation of BMMSCs induced by TGC-CMM, in accordance, MagT1 knockdown significantly decreased the extent of mineralized nodules and the protein levels of alkaline phosphatase (ALP), dentin matrix protein 1 (DMP-1), and dentin sialophosphoprotein (DSP). Flow cytometry showed that intracellular Mg2+ was significantly reduced in MagT1-knockdown BMMSCs, indicating the suppression of MagT1 inhibited odontogenic differentiation of BMMSCs by decreasing intracellular Mg2+. Finally, we performed RNA-seq to explore the altered transcriptome of MagT1-knockdown BMMSCs undergoing odontogenic differentiation and identified 281 differentially expressed genes, some of which were involved in MAPK pathway. Consistently, automated western blot analysis found the ERK/MAPK pathway was inhibited in MagT1-knockdown BMMSCs during odontogenic differentiation, indicating that suppression of MagT1 inhibited odontogenic differentiation of BMMSCs via ERK/MAPK pathway. CONCLUSIONS: This study identified the significant alteration of transcriptome in BMMSCs undergoing odontogenic differentiation induced by TGC-CM. We clarified the pivotal role of MagT1 and ERK/MAPK pathway in odontogenic differentiation of BMMSCs, and suppression of MagT1 inhibited the odontogenic differentiation of BMMSCs by decreasing the intracellular Mg2+ and inactivating ERK/MAPK pathway.

Cancer-associated fibroblasts contribute to oral cancer cells proliferation and metastasis via exosome-mediated paracrine miR-34a-5p.

BACKGROUND: Cancer-associated fibroblasts (CAFs) play an important role in regulating tumor progression by transferring exosomes to neighboring cells. Our aim was to clarify the role of microRNA encapsulated in the exosomes derived from CAFs in oral squamous cell carcinoma (OSCC). METHODS: We examined the microRNA expression profiles of exosomes derived from CAFs and donor-matched normal fibroblasts (NFs) from patients with OSCC. We used confocal microscopy to examine the transportation of exosomal miR-34a-5p between CAFs and OSCC cells. Next, luciferase reporter and its mutant plasmids were used to confirm direct target gene of miR-34a-5p. Phenotypic assays and in vivo tumor growth experiments were used to investigate the functional significance of exosomal miR-34a-5p. FINDINGS: We found that the expression of miR-34a-5p in CAF-derived exosomes was significantly reduced, and fibroblasts could transfer exosomal miR-34a-5p to OSCC cells. In xenograft experiments, miR-34a-5p overexpression in CAFs could inhibit the tumorigenesis of OSCC cells. We further revealed that miR-34a-5p binds to its direct downstream target AXL to suppress OSCC cell proliferation and metastasis. Stable ectopic expression of AXL in OSCC cells overexpressing miR-34a-5p restored proliferation and motility abolished by the miRNA. The miR-34a-5p/AXL axis promoted OSCC progression via the AKT/GSK-3ß/ß-catenin signaling pathway, which could induce the epithelial-mesenchymal transition (EMT) to promote cancer cells metastasis. The miR-34a-5p/AXL axis enhanced nuclear translocation of ß-catenin and then induced transcriptional upregulation of SNAIL, which in turn activated both MMP-2 and MMP-9. INTERPRETATION: The miR-34a-5p/AXL axis confers aggressiveness in oral cancer cells through the AKT/GSK-3ß/ß-catenin/Snail signaling cascade and might represent a therapeutic target for OSCC. FUND: National Natural Science Foundation of China.

Foxc2 and BMP2 Induce Osteogenic/Odontogenic Differentiation and Mineralization of Human Stem Cells from Apical Papilla.

As a transcription factor regulated by bone morphogenetic protein 2 (BMP2), Forkhead c2 (Foxc2) plays a pivot role in osteogenesis/odontogenesis. However, the role of Foxc2 and BMP2 in regulating osteo-/odontogenic differentiation and mineralization of stem cells from apical papilla (SCAP) is still uncertain. In this research, overexpression of Foxc2 gene significantly improved the proliferation of SCAP four days and eight days after transfection, but overexpression of both Foxc2 and BMP2 genes significantly inhibited the proliferation of SCAP eight days after transfection. RT-qPCR and western blot results indicated that SCAP-Foxc2-BMP2 significantly upregulated osteo-/odontogenic genes and proteins at most of the time points in SCAP after transfection. Moreover, SCAP-Foxc2-BMP2 formed notably more alkaline phosphatase-positive and alizarin red-positive mineralized nodules than other three group cells sixteen days after transfection. In conclusion, our findings revealed that Foxc2 and BMP2 synergistically promoted osteo-/odontogenic differentiation and mineralization of SCAP in vitro.

The relationship between vitamin D receptor gene polymorphism and deciduous tooth decay in Chinese children.

BACKGROUND: In the present study, we explored the link between vitamin D receptor (VDR) BsmI, TaqI, ApaI and FokI gene polymorphisms with deciduous tooth decay in Chinese children. METHODS: Our study included 380 Chinese children aged 4-7 years, whose DNA sample was collected from the buccal mucosa. VDR gene polymorphisms was determined by PCR-RFLP. RESULTS: The adjusted logistic regression analysis demonstrated that BsmI containing the Bb genotype was linked with the increased risk of deciduous tooth decay (OR = 1.856, 95% CI = [1.184, 2.908], p = 0.007). However, VDR polymorphisms ApaI, TaqI and FokI were not associated with deciduous tooth decay (ApaI: OR = 0.839, 95% CI = [0.614, 1.145], p = 0.268; TaqI: OR = 1.150, 95% CI = [0.495, 2.672], p = 0.744; FokI: OR = 0.856, 95% CI = [0.616, 1.191], p = 0.356). CONCLUSIONS: Our results showed that VDR BsmI polymorphism was associated with the risk of deciduous tooth decay in Chinese children aged 4-7 years. However, the specific mechanism remains to further verify through experiment.

Role of Magnesium Transporter Subtype 1 (MagT1) in the Osteogenic Differentiation of Rat Bone Marrow Stem Cells.

In the present study, we investigated the role of magnesium transporter subtype 1 (MagT1), a selective Mg transporter protein, in the osteogenic differentiation of rat bone marrow stem cells (rBMSCs). Osteogenic differentiation was monitored by the expressions of alkaline phosphatase (ALP), osteocalcin (OCN), collagen-1 (COL-1) and runt-related transcription factor 2 (RUNX2), and extracellular matrix mineralization of rBMSCs. The expression of MagT1 increased with osteogenic differentiation of rBMSCs, suggesting the importance of intracellular Mg homeostasis to cell differentiation. Alteration of intracellular Mg homeostasis by culture condition with low extracellular Mg significantly reduced the osteogenic differentiation markers ALP, OCN, COL-1, and RUNX2 gene expressions. MagT1 knockdown during the differentiation period also reduced osteogenic differentiation and the extent of matrix mineralization of rBMSCs. In conclusion, our results indicate that Mg and MagT1 play an important role in osteogenic differentiation of rBMSCs and may be involved in the bone regeneration.

Proliferation and odontogenic differentiation of BMP2 gene­transfected stem cells from human tooth apical papilla: an in vitro study.

Stem cells from the apical papilla (SCAP) have odontogenic potential, which plays a pivotal role in the root dentin development of permanent teeth. Human bone morphogenetic protein 2 (BMP2) is a well-known gene that participates in regulating the odontogenic differentiation of dental tissue­derived stem cells. However, little is known regarding the effects of the BMP2 gene on the proliferation and odontogenic differentiation of SCAP. This study aimed to evaluate the odontogenic differentiation potential of lentiviral­mediated BMP2 gene­transfected human SCAP (SCAP/BMP2) in vitro. SCAP were isolated by enzymatic dissociation of human teeth apical papillae. The multipotential of SCAP was verified by their osteogenic and adipogenic differentiation characteristics. The phenotype of SCAP was evaluated by flow cytometry (FCM). The proliferation status of the blank vector­transfected SCAP (SCAP/Vector) and SCAP/BMP2 was analyzed by a cell counting kit-8 (CCK­8). Odontogenic genes, including alkaline phosphatase (ALP), osteocalcin (OCN), dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1) of the two groups of cells were evaluated by quantitative polymerase chain reaction (qPCR). ALP staining and alizarin red (AR) staining of the cells was performed on the 16th day after transfection. In vitro results of CCK-8, qPCR, ALP and AR staining demonstrated that: ⅰ) SCAP/BMP2 had a comparable proliferation rate to SCAP/Vector; ⅱ) SCAP/BMP2 presented significantly better potential to differentiate into odontoblasts compared to SCAP/Vector by upregulating ALP, OCN, DSPP and DMP1 genes; ⅲ) more ALP granules and mineralized deposits were formed by SCAP/BMP2 as compared to SCAP/Vector. The results suggested that lentiviral-mediated BMP2 gene transfection enhances the odontogenic differentiation capacity of human SCAP in vitro.

Low serum levels of zinc, copper, and iron as risk factors for osteoporosis: a meta-analysis.

Zinc (Zn), copper (Cu), and iron (Fe) are essential trace elements for the growth, development, and maintenance of healthy bones. However, there are conflicting reports as to the relationship between serum level of Zn, Cu, or Fe and osteoporosis (OP). The purpose of the present study is to clarify the relationship between serum Zn, Cu, or Fe and OP using a meta-analysis approach. We searched all articles indexed in PubMed published up to May 2014 concerning the association between serum level of Zn, Cu, or Fe and OP. Eight eligible articles involving 2,188 subjects were identified. Overall, pooled analysis indicated that patients with OP had a lower serum level of Zn, Cu, or Fe than the healthy controls (Zn standardized mean difference (SMD) = -1.396, 95% confidence interval (CI) = [-2.129, -0.663]; Cu SMD = -0.386, 95% CI = [-0.538, -0.234]; Fe SMD = -0.22, 95% CI = [-0.30, -0.13]). Further subgroup analysis found that geographical location and gender had an influence on the serum level of Zn in OP and healthy controls, but not on the serum level of Cu or Fe. No evidence of publication bias was observed. In conclusion, this meta-analysis suggests that low serum levels of Zn, Cu, and Fe seem to be important risk factors for OP and well-designed studies with adequate control for confounding factors are required in future investigations.

Association between serum level of magnesium and postmenopausal osteoporosis: a meta-analysis.

There are conflicting reports as to the association between serum level of magnesium (Mg) and postmenopausal osteoporosis (OP). The purpose of the present study is to clarify the association between serum level of Mg and postmenopausal OP using a meta-analysis approach. We searched articles indexed in Pubmed and the Chinese Journal Full-text Database (CJFD) published as of October 2013 that met our predefined criteria. Seven eligible studies involving 1,349 postmenopausal women from 12 case-control study arms were identified. Overall, pooled analysis indicated that postmenopausal osteoporotic women had a lower serum level of Mg than the healthy controls (standardized mean difference [SMD]=-0.55, 95 % confidence interval [CI]=-0.83 to -0.26). Further subgroup analysis found a similar pattern in Turkey (SMD=-0.66, 95% CI=-0.99 to -0.32) and Belgium (SMD=-0.98, 95% CI=-1.91 to -0.05), but not in China (SMD=0.02, 95% CI=-0.21 to 0.26). And the difference of serum level of Mg between postmenopausal osteoporotic women and healthy controls below the age of 60 years (SMD=-0.61, 95% CI=-1.09 to -0.13) was similar to that among the population over 60 years (SMD=-0.49, 95% CI=-0.80 to -0.18).In conclusion, this meta-analysis suggests that the low serum level of Mg seems to be a risk factor for OP among the postmenopausal women. However, the subgroup analysis found that there was contradiction regarding races and geography, like China and Turkey. Thus, this finding needs further confirmation by trans-regional multicenter study to obtain better understanding of causal relationships between serum Mg and postmenopausal OP.