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Tooth ankylosis is a pathological condition of periodontal ligament (PDL) restoration after tooth replantation. Platelet-derived growth factor-BB (PDGF-BB) has been proposed as a promising factor for preventing tooth ankylosis. Using rat tooth replantation model, we investigated whether PDGF-BB accelerates the repair of PDL after tooth replantation without ankylosis, and its molecular mechanisms. In PDGF-BB pretreated replanted teeth (PDGF-BB group), ankylosis was markedly reduced and functionally organized PDL collagen fibers were restored; the mechanical strength of the healing PDL was restored to an average of 76% of that in non-replanted normal teeth at 21 days. The numbers of PDGF-Rβ- and BrdU-positive cells in the periodontal tissues of the PDGF-BB group were greater than those of atelocollagen pretreated replanted teeth (AC group). Moreover, in the PDGF-BB group, the periodontal tissues had fewer osteocalcin-positive cells and decreased number of nuclear β-catenin-positive cells compared to those in the AC group. In vitro analyses showed that PDGF-BB increased the proliferation and migration of human periodontal fibroblasts. PDGF-BB downregulated mRNA expressions of RUNX2 and ALP, and inhibited upregulatory effects of Wnt3a on β-catenin, AXIN2, RUNX2, COL1A1, and ALP mRNA expressions. These findings indicate that in tooth replantation, topical PDGF-BB treatment enhances cell proliferation and migration, and inhibits canonical Wnt signaling activation in bone-tooth ankylosis, leading to occlusal loading of the PDL tissues and subsequent functional restoration of the healing PDL. This suggests a possible clinical application of PDGF-BB to reduce ankylosis after tooth replantation and promote proper regeneration of PDL.

G9a is a histone methyltransferase that catalyzes the methylation of histone 3 lysine 9 (H3K9), which is involved in the regulation of gene expression. We had previously reported that G9a is expressed in developing tendons in vivo and in vitro and that G9a-deficient tenocytes show impaired proliferation and differentiation in vitro. In this study, we investigated the functions of G9a in tendon development in vivo by using G9a conditional knockout ( G9a cKO) mice. We crossed Sox9 Cre/ + mice with G9a fl/fl mice to generate G9a fl/fl ; Sox9 Cre/ + mice. The G9a cKO mice showed hypoplastic tendon formation at 3 weeks of age. Bromodeoxyuridine labeling on embryonic day 16.5 (E16.5) revealed decreased cell proliferation in the tenocytes of G9a cKO mice. Immunohistochemical analysis revealed decreased expression levels of G9a and its substrate, H3K9me2, in the vertebral tendons of G9a cKO mice. The tendon tissue of the vertebrae and limbs of G9a cKO mice showed reduced expression of a tendon marker, tenomodulin ( Tnmd ), and col1a1 genes, suggesting that tenocyte differentiation was suppressed. Overexpression of G9a resulted in enhancement of Tnmd and col1a1 expression in tenocytes in vitro. These results suggest that G9a regulates the proliferation and differentiation of tendon progenitor cells during tendon development. Thus, our results suggest that G9a plays an essential role in tendon development.

Development of regenerative therapies for damaged tendons remains a great challenge, largely because of lack of information regarding the mechanisms responsible for differentiation of tenocytes. Mouse tenocytes have not been fully characterized owing to the absence of efficient and reproducible methods for their in vitro expansion without losing phenotypic features. The objective of the study was to establish an improved and reliable method for stable primary culture of mouse tenocytes by using collagen gel. Achilles and tail tendon tissues were harvested and embedded in collagen gel. After 10 days of continuous culture, the gel was digested and cells were passaged on tissue culture-treated plastic dishes. Mouse tenocytes cultured in collagen gel exhibited significantly shorter doubling time and higher numbers of proliferation when maintained on the plastic dishes compared with those cultured without using gel. Transmission electron microscopic analyses showed that cultured tenocytes retained some morphological features of tenocytes in tendon tissues, such as cell–cell junctional complex formation, well-developed rough endoplasmic reticulum, and mitochondria in their cytoplasm. mRNA expression of tenocyte markers (tenomodulin, type I collagen, periostin, and scleraxis) was higher in cells cultured in collagen gel than in those cultured in the absence of gel. Our results show that tenocytes cultured using the collagen gel method express typical lineage markers and exhibit improved growth characteristics, thus providing a stable platform for studying molecular mechanisms that control their differentiation.

The β-galactosidase gene ( lacZ ) of Escherichia coli is widely used as a reporter gene. The expression of lacZ can be detected by enzyme-based histochemical staining using chromogenic substrates such as 5-bromo-4-chloro-3-indolyl-β- d -galactoside (X-gal). Because the enzymatic activity of lacZ is vulnerable to high temperatures and acid treatment for demineralization, detection of lacZ on paraffinized sections is difficult, especially for hard tissues, which require demineralization before sectioning in paraffin. To circumvent this problem, whole-mount X-gal staining before sectioning is performed. However, detection of lacZ activity in the center of larger portions of hard whole adult tissues is challenging. In this study, focusing on fixation procedures, we determined the conditions conducive to improved detection of lacZ activity in deeper areas of whole tissues. We used an annexin a5 ( Anxa5 )- lacZ reporter mouse model in which the Anxa5 expression in hard tissue is indicated by lacZ activity. We found that lacZ activity could be detected throughout the periodontal ligament of adult mice when fixed in 100% acetone, whereas it was not detected in the periodontal ligament around the root apex fixed in glutaraldehyde and paraformaldehyde. This staining could not be detected in wild-type mice. Acetone maintains the lacZ activity within 48 h of fixation at both 4°C and at room temperature. In conclusion, acetone is the optimal fixative to improve permeability for staining of lacZ activity in large volumes of adult hard tissues.

The β-galactosidase gene (lacZ) of Escherichia coli is widely used as a reporter gene. The expression of lacZ can be detected by enzyme-based histochemical staining using chromogenic substrates such as 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-gal). Because the enzymatic activity of lacZ is vulnerable to high temperatures and acid treatment for demineralization, detection of lacZ on paraffinized sections is difficult, especially for hard tissues, which require demineralization before sectioning in paraffin. To circumvent this problem, whole-mount X-gal staining before sectioning is performed. However, detection of lacZ activity in the center of larger portions of hard whole adult tissues is challenging. In this study, focusing on fixation procedures, we determined the conditions conducive to improved detection of lacZ activity in deeper areas of whole tissues. We used an annexin a5 (Anxa5)-lacZ reporter mouse model in which the Anxa5 expression in hard tissue is indicated by lacZ activity. We found that lacZ activity could be detected throughout the periodontal ligament of adult mice when fixed in 100% acetone, whereas it was not detected in the periodontal ligament around the root apex fixed in glutaraldehyde and paraformaldehyde. This staining could not be detected in wild-type mice. Acetone maintains the lacZ activity within 48 h of fixation at both 4°C and at room temperature. In conclusion, acetone is the optimal fixative to improve permeability for staining of lacZ activity in large volumes of adult hard tissues.[PUBLICATION ABSTRACT]

The aims of this study are to observe microscopic changes in the periodontal ligament (PDL) collagen fibres after collagenase treatment, to analyse stress–relaxation behaviour of PDL specimens treated with collagenase, and to elucidate the contribution of the collagen component to the viscoelastic behaviour of the PDL. Transverse sections of rat mandibular first molars ( n=24) were treated in vitro with 0, 8, 16, or 24 units of bacterial collagenase for 4 h at 37 °C. Histological specimens were then prepared, and image analyses were done for polarised light microscopic appearances of collagen fibres. Further, stress–relaxation tests were performed for PDL specimens treated with 8 units of collagenase ( n=7) and control specimens ( n=7). Image analysis showed that higher concentrations of collagenase reduced greater area occupied by the PDL collagen fibres and birefringent retardation of the fibres. The amount of stress–relaxation during 600 s was 1.37 times greater in the collagenase-treated specimens than in the controls. The observed values of the stress–relaxation process were well described by a function with three exponential decay terms. The relaxation parameters of the first and second terms did not show significant differences, but those of the third term did so between the collagenase-treated and control specimens. The ratio and relaxation time of the third term for the collagenase-treated specimens were significantly less than those for the controls. These findings suggest that in the long-term relaxation component of the stress–relaxation process of the PDL the viscoelastic properties of the collagen fibres may play an important role.

In this paper, we report ecological and environmental investigations on Pteris vittata in the As–Pb–Hg-polluted Bone River area, Gorontalo Province, Indonesia. The density distribution of P. vittata decreases from around the artisanal and small-scale gold mining (ASGM) site to the lower reaches of the Bone River, and it is rarely found near Gorontalo City. The maximum concentrations of As, Hg, and Pb recorded in the soil samples were 401, 36, and 159 mg kg−1, respectively, with their maximum concentrations in P. vittata recorded as 17,700, 5.2, and 39 mg kg−1, respectively. Around the ASGM sites, the concentrations of As, Pb, and Hg in P. vittata were highest in the study area. These data suggest that P. vittata, a hyperaccumulator of As, may be useful as a bioindicator for assessing environmental pollution by Pb and Hg.

Esophageal squamous cell carcinoma (ESCC) is an intractable digestive organ cancer that has proven difficult to treat despite multidisciplinary therapy, and a new treatment strategy is demanded. Metformin is used for type 2 diabetes mellitus and its antitumor effects have been reported recently. Metformin exerts antitumor effects in various respects, such as inhibiting inflammation, tumor growth and epithelial‐mesenchymal transition (EMT). However, few reports have described the efficacy of metformin on ESCC, and their findings have been controversial. We analyzed the antitumor effects of metformin and clarified its effects on anti‐inflammation, growth suppression and EMT inhibition. Activation of nuclear factor kappa B (NF‐κB), the major transcription factor induced by inflammation, was investigated by immunostaining. We found that localization of NF‐κB in the nucleus was reduced after metformin treatment. This suggests that metformin inhibited the activation of NF‐κB. Metformin inhibited tumor growth and induced apoptosis in ESCC cell lines. Associated with EMT, we examined cell motility by a wound healing assay and the epithelial marker E‐cadherin expression of various ESCC cell lines by western blotting. Metformin inhibited cell motility and induced E‐cadherin expression. In conclusion, metformin showed multiple antitumor effects such as growth suppression, invasion inhibition, and control of EMT by inhibiting NF‐κB localization on ESCC. Further exploration of the marker of treatment efficacy and combination therapy could result in the possibility for novel treatment to use metformin on ESCC. Metformin, which is used for type 2 diabetes mellitus, showed antitumor effects by inhibiting cell proliferation, tumor growth and EMT in esophageal squamous cell carcinoma. These effects might have been induced by inhibiting NF‐κB activation.

Background The relationship between oral anticoagulant (OAC) status after catheter ablation (CA) for atrial fibrillation (AF) and the risks of ischemic stroke or major bleeding events is still unknown. Methods This is a subanalysis of the RYOUMA registry, a prospective multicenter observational study of Japanese patients who underwent CA for AF in 2017–2018. Results Of the 2844 patients, the rate of DOAC continuation was 48.1%, 69.6%, and 80.9% in patients with a CHADS2 score of 0–1, 2, and 3–6, respectively. Among the patients taking DOACs with a CHADS2 score of 0–1 and 2, the incidence rates of major bleeding were significantly higher than those of ischemic stroke or systemic embolic events (SEEs) (1.3%/year [95% CI, 0.6–2.1] vs. 0.3%/year [95% CI, 0.0–0.7], p = 0.019; 1.8%/year [95% CI, 0.6–3.0] vs. 0.2%/year [95% CI, 0.0–0.6], p = 0.018, respectively). However, there was no difference between the incidence rates of major bleeding events and ischemic stroke or SEEs in patients taking DOACs with a CHADS2 score of 3–6 (1.6%/year [95% CI, 0.2–3.0] vs. 1.0%/year [95% CI, 0.0–2.1], p = 0.474). Conclusions In patients with a CHADS2 score of 2, those who continued taking DOACs had a higher incidence rate of major bleeding events compared to ischemic stroke/SEEs, similar to those with a CHADS2 score of 0–1. Conversely, in patients with a CHADS2 score of 3–6, the incidence rates of both ischemic stroke/SEEs and major bleeding were similarly high. Trial Registration: The study was registered as UMIN000026092 (University Hospital Medical Information Network‐Clinical Trial Registry)