625 nm Light Irradiation Prevented MC3T3-E1 Cells from Accumulation of Misfolded Proteins via ROS and ATP Production.

Osteoblasts must acquire a considerable capacity for folding unfolded and misfolded proteins (MPs) to produce large amounts of extracellular matrix proteins and maintain bone homeostasis. MP accumulation contributes to cellular apoptosis and bone disorders. Photobiomodulation therapy has been used to treat bone diseases, but the effects of decreasing MPs with photobiomodulation remain unclear. In this study, we explored the efficacy of 625 nm light-emitting diode irradiation (LEDI) to reduce MPs in tunicamycin (TM) induced-MC3T3-E1 cells. Binding immunoglobulin protein (BiP), an adenosine triphosphate (ATP)-dependent chaperone, is used to evaluate the capacity of folding MPs. The results revealed that pretreatment with 625 nm LEDI (Pre-IR) induced reactive oxygen species (ROS) production, leading to the increased chaperone BiP through the inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1s (XBP-1s) pathway, and then restoration of collagen type I (COL-I) and osteopontin (OPN) expression relieving cell apoptosis. Furthermore, the translocation of BiP into the endoplasmic reticulum (ER) lumen might be followed by a high level of ATP production. Taken together, these results suggest that Pre-IR could be beneficial to prevent MP accumulation through ROS and ATP in TM-induced MC3T3-E1cells.

Effects of Red LED Irradiation in Enhancing the Mineralization of Human Dental Pulp Cells In Vitro.

Dentin regeneration is the preferred method used to preserve dental pulp vitality after pulp exposure due to caries. Red light-emitting diode irradiation (LEDI), which is based on photobiomodulation (PBM), has been used to promote hard-tissue regeneration. However, the underlying mechanism still needs elucidation. This study aimed to explore the mechanism involved in red LEDI affecting dentin regeneration. Alizarin red S (ARS) staining revealed that red LEDI induced mineralization of human dental pulp cells (HDPCs) in vitro. We further distinguished the cell proliferation (0-6 d), differentiation (6-12 d), and mineralization (12-18 d) of HDPCs in vitro and treated cells either with or without red LEDI in each stage. The results showed that red LEDI treatment in the mineralization stage, but not the proliferation or differentiation stages, increased mineralized nodule formation around HDPCs. Western blot also indicated that red LEDI treatment in the mineralization stage, but not the proliferation or differentiation stages, upregulated the expression of dentin matrix marker proteins (dentin sialophosphoprotein, DSPP; dentin matrix protein 1, DMP1; osteopontin, OPN) and an intracellular secretory vesicle marker protein (lysosomal-associated membrane protein 1, LAMP1). Therefore, the red LEDI might enhance the matrix vesicle secretion of HDPCs. On the molecular level, red LEDI enhanced mineralization by activating the mitogen-activated protein kinase (MAPK) signaling pathways (ERK and P38). ERK and P38 inhibition reduced mineralized nodule formation and the expression of relevant marker proteins. In summary, red LEDI enhanced the mineralization of HDPCs by functioning to produce a positive effect in the mineralization stage in vitro.

Pirfenidone Inhibits Alveolar Bone Loss in Ligature-Induced Periodontitis by Suppressing the NF-κB Signaling Pathway in Mice.

There has been increasing interest in adjunctive use of anti-inflammatory drugs to control periodontitis. This study was performed to examine the effects of pirfenidone (PFD) on alveolar bone loss in ligature-induced periodontitis in mice and identify the relevant mechanisms. Experimental periodontitis was established by ligating the unilateral maxillary second molar for 7 days in mice (n = 8 per group), and PFD was administered daily via intraperitoneal injection. The micro-computed tomography and histology analyses were performed to determine changes in the alveolar bone following the PFD administration. For in vitro analysis, bone marrow macrophages (BMMs) were isolated from mice and cultured with PFD in the presence of RANKL or LPS. The effectiveness of PFD on osteoclastogenesis, inflammatory cytokine expression, and NF-κB activation was determined with RT-PCR, Western blot, and immunofluorescence analyses. PFD treatment significantly inhibited the ligature-induced alveolar bone loss, with decreases in TRAP-positive osteoclasts and expression of inflammatory cytokines in mice. In cultured BMM cells, PFD also inhibited RANKL-induced osteoclast differentiation and LPS-induced proinflammatory cytokine (IL-1ß, IL-6, TNF-a) expression via suppressing the NF-κB signal pathway. These results suggest that PFD can suppress periodontitis progression by inhibiting osteoclastogenesis and inflammatory cytokine production via inhibiting the NF-κB signal pathway, and it may be a promising candidate for controlling periodontitis.

Esthetic reconstruction of a localized severely resorbed anterior maxilla associated with peri-implantitis: A clinical report.

This clinical report described the esthetic reconstruction of a localized severely resorbed right anterior maxilla associated with peri-implantitis. For vertical bone augmentation, guided bone regeneration surgery was performed by raising a flap with the remote incision technique, followed by soft tissue grafting and vestibuloplasty. The biologically oriented preparation technique was used to improve the health and stability of the peri-implant tissues. The surgical treatment and a novel method of prosthetic rehabilitation provided excellent esthetic and functional outcomes.

Body mass index and self-rated oral health in Korean adults in 2017.

OBJECTIVE: To investigate the association between obesity and self-rated oral health (SROH). This study examined the cross-sectional associations between body mass index (BMI) and SROH in Korean adults. MATERIALS AND METHODS: This study used data from 217 304 adults (100 110 men and 117 194 women aged > 19 years) from the 2017 Korean Community Health Survey. Participants were categorised into six ordinal groups based on BMI: underweight (<18.5 kg/m2 ), normal weight (18.5-22.9 kg/m2 ), overweight (23.0-24.9 kg/m2 ), obese-I (25.0-27.4 kg/m2 ), obese-II (27.5-29.9 kg/m2 ) or obese-III (≥30.0 kg/m2 ). SROH was assessed using responses to the question, "How do you rate your oral health, including your teeth and gums?" rated on a 5-point scale. SROH was categorised as "good" (reported as "fair," "good" or "very good") or "poor" or "very poor." Age- and sex-stratified associations between BMI categories and poor SROH were assessed using ordinal logistic regression analysis with sampling weights. RESULTS: The age-adjusted odds ratio (OR) for poor SROH according to BMI levels was lowest in the overweight group in both men and women. In men, the OR for poor SROH was 2.03 (99% confidence interval [CI], 1.72-2.39) in the underweight group, 1.17 (99% CI, 1.17-1.25) in the normal group, 1.05 (99% CI, 0.98-1.13) in the obese-I group, 1.08 (99% CI, 0.98-1.18) in the obese-II group and 1.36 (99% CI, 1.20-1.55) in the obese-III group. In women, the OR was 1.18 (99% CI, 1.07-1.31) in the underweight group, 1.01 (99% CI, 0.95-1.07) in the normal group, 1.07(99% CI, 0.99-1.16) in the obese-I group, 1.16 (99% CI, 1.04-1.30) in the obese-II group and 1.39 (99% CI, 1.20-1.62) in the obese-III group. From the restricted cubic spline models in both sexes, BMI showed a J-shaped association with poor and very poor SROH in men and women. In a stratified analysis by age group and sex, men and older women in the underweight group had poorer SROH than those in overweight group. CONCLUSION: In a nationally representative sample of Korean adults, there was a J-shaped association between BMI and poor SROH, with the highest risk in the underweight group amongst men and in the obese-III group amongst women. Furthermore, in men and women over 65 years of age, underweight and obesity were associated with poorer SROH.

Activation and increased production of interleukin-17 and tumour necrosis factor-α of mucosal-associated invariant T cells in patients with periodontitis.

AIM: Mucosal-associated invariant T (MAIT) cells are known to be resident in oral mucosal tissue, but their roles in periodontitis are unknown. This study aimed to examine the level and function of MAIT cells in periodontitis patients. MATERIALS AND METHODS: Frequency, activation, and function of MAIT cells from 28 periodontitis patients and 28 healthy controls (HCs) were measured by flow cytometry. RESULTS: Circulating MAIT cells were numerically reduced in periodontitis patients. Moreover, they exhibited higher expression of CD69 and annexin V, together with more increased production of interleukin (IL)-17 and tumour necrosis factor (TNF)-α, in periodontitis patients than in HCs. Interestingly, periodontitis patients had higher frequencies of MAIT cells in gingival tissue than in peripheral blood. In addition, circulating MAIT cells had elevated expression of tissue-homing chemokine receptors such as CCR6 and CXCR6, and the corresponding chemokines (i.e., CCL20 and CXCL16) were more strongly expressed in inflamed gingiva than in healthy gingiva. CONCLUSIONS: This study demonstrates that circulating MAIT cells are numerically deficient with an activated profile toward the production of IL-17 and TNF-α in periodontitis patients. Furthermore, circulating MAIT cells have the potential to migrate to inflamed gingival tissues.

Sclerostin in periodontal ligament: Homeostatic regulator in biophysical force-induced tooth movement.

AIM: To study the role of sclerostin in periodontal ligament (PDL) as a homeostatic regulator in biophysical-force-induced tooth movement (BFTM). MATERIALS AND METHODS: BFTM was performed in rats, followed by microarray, immunofluorescence, in situ hybridization, and real-time polymerase chain reaction for the detection and identification of the molecules. The periodontal space was analysed via micro-computed tomography. Effects on osteoclastogenesis and bone resorption were evaluated in the bone-marrow-derived cells in mice. In vitro human PDL cells were subjected to biophysical forces. RESULTS: In the absence of BFTM, sclerostin was hardly detected in the periodontium except in the PDL and alveolar bone in the furcation region and apex of the molar roots. However, sclerostin was up-regulated in the PDL in vivo by adaptable force, which induced typical transfiguration without changes in periodontal space as well as in vitro PDL cells under compression and tension. In contrast, the sclerostin level was unaffected by heavy force, which caused severe degeneration of the PDL and narrowed periodontal space. Sclerostin inhibited osteoclastogenesis and bone resorption, which corroborates the accelerated tooth movement by the heavy force. CONCLUSIONS: Sclerostin in PDL may be a key homeostatic molecule in the periodontium and a biological target for the therapeutic modulation of BFTM.

SLPI in periodontal Ligament is not sleepy during biophysical force-induced tooth movement.

AIM: We aimed to identify a key molecule that maintains periodontal tissue homeostasis during biophysical force-induced tooth movement (BTM) by orchestrating alveolar bone (AB) remodelling. MATERIALS AND METHODS: Differential display-PCR was performed to identify key molecules for BTM in rats. To investigate the localization and expression of the identified molecules, immunofluorescence, real-time RT-PCR and Western blotting were performed in rats and human periodontal ligament (PDL) cells. Functional test and micro-CT analysis were performed to examine the in vivo effects of the identified molecules on BTM. RESULTS: Secretory leucocyte peptidase inhibitor (SLPI) in the PDL was revealed as a key molecule for BTM-induced AB remodelling. SLPI was enhanced in the PDL under both compression and tension, and downregulated by an adenyl cyclases inhibitor. SLPI induced osteoblastogenic genes including runt-related transcription factor 2 (Runx2) and synergistically augmented tension-induced Runx2 expression. SLPI augmented mineralization in PDL cells. SLPI induced osteoclastogenic genes including receptor activator of nuclear factor kappa-Β ligand (RANKL) and synergistically augmented the compression-induced RANKL and macrophage colony-stimulating factor (MCSF) expression. Finally, the in vivo SLPI application into the AB significantly augmented BTM. CONCLUSIONS: SLPI or its inhibitors might serve as a biological target molecule for therapeutic interventions to modulate BTM.

The Protective Role of Feruloylserotonin in LPS-Induced HaCaT Cells.

Epidermal inflammation is caused by various bacterial infectious diseases that impair the skin health. Feruloylserotonin (FS) belongs to the hydroxycinnamic acid amides of serotonin, which mainly exists in safflower seeds and has been proven to have anti-inflammatory and antioxidant activities. Human epidermis mainly comprises keratinocytes whose inflammation causes skin problems. This study investigated the protective effects of FS on the keratinocyte with lipopolysaccharides (LPS)-induced human HaCaT cells and elucidated its underlying mechanisms of action. The mechanism was investigated by analyzing cell viability, PGE2 levels, cell apoptosis, nuclear factor erythroid 2-related factor 2 (Nrf2) translocation, and TLR4/NF-κB pathway. The anti-inflammatory effects of FS were assessed by inhibiting the inflammation via down-regulating the TLR4/NF-κB pathway. Additionally, FS promoted Nrf2 translocation to the nucleus, indicating that FS showed anti-oxidative activities. Furthermore, the antioxidative and anti-inflammatory effects of FS were found to benefit each other, but were independent. Thus, FS can be used as a component to manage epidermal inflammation due to its anti-inflammatory and anti-oxidative properties.

Digital panoramic radiographs are useful for diagnosis of osteoporosis in Korean postmenopausal women.

OBJECTIVE: The purpose of this study was to determine whether digital panoramic radiographs could be used for the diagnosis of osteoporosis through evaluation of the radiographs based on the correlation with bone mineral density (BMD). METHODS: One hundred and ninety-four post-menopausal women were selected from participants who had participated in the Dong-gu study. Panoramic radiographic indices measured are mental index (MI), mandibular cortical index (MCI) and simple visual estimation (SVE). BMD at the lumbar spine and proximal femur was measured by dual-energy X-ray absorptiometry (DXA). The Pearson's correlation test was performed to analyse the correlation between MI and age and BMD at the lumbar spine, femoral neck and total hip. Multiple linear regression analysis was performed to analyse the association of MI, MCI and SVE with BMD after adjusting for age, height and weight. To determine the optimal cut-off point of MI for the diagnosis of osteoporosis, the receiver operating characteristic analysis was performed. RESULTS: The MI was positively correlated with BMDs: lumbar spine: r = 0.36, femoral neck: r = 0.59 and total hip: r = 0.58 (p < 0.001). As age increased, MI decreased (r = -0.46). BMD at the lumbar spine and total hip were significantly lower in participants with reduction of mandibular width, thinning and resorption of mandibular cortex by the MI, SVE and MCI, respectively. The optimal cut-off value of MI for the diagnosis of spinal osteoporosis was 2.22 mm. CONCLUSION: Thickness and morphological changes of mandibular inferior cortical bone are associated with BMD, independent of age, height and weight. These results suggest that MI, MCI and SVE may be useful indices for the diagnosis of osteoporosis in a Korean population.

Periodontal disease associated with blood glucose levels in urban Koreans aged 50 years and older: the Dong-gu study.

OBJECTIVE: To investigate the association of periodontal disease and the number of teeth present with the risk of prediabetes and diabetes as well as with blood glucose and HbA1c levels in adult Koreans. BACKGROUND: The relationship between periodontal disease and diabetes has not been fully elucidated. MATERIALS AND METHODS: Cross-sectional data from 5535 participants aged ≥50 years were obtained from 2008 to 2010. Periodontal status was measured as pocket depth (PD), clinical attachment loss (CAL) and bleeding on probing (BOP) recorded. The percentage of sites with a PD ≥4 mm, CAL ≥4 mm (CAL4) and BOP (BOP%) were recorded. Participants were divided into three groups according to PD4, CAL4 and BOP% measurements. Number of teeth present was divided into four groups. Participants were classified as normoglycaemic, prediabetic or diabetic based on HbA1c and fasting glucose levels. RESULTS: After full adjustment, the highest tertile of CAL4 (OR: 1.47, 95% CI: 1.18-2.02, p < 0.001), PD4 (OR: 1.58, 95% CI: 1.26-1.97, p < 0.001) and BOP% (OR: 1.37, 95% CI: 1.07-1.75, p = 0.012) had significantly increased odds of diabetes. The number of teeth present was inversely related to diabetes (p < 0.001) and prediabetes (p = 0.032) risk. Periodontal disease severity was positively associated with HbA1c and glucose levels. The number of teeth present was positively associated with HbA1c, but not glucose, levels. CONCLUSION: Periodontal disease and the number of teeth present are associated with an increased risk of diabetes and increased blood glucose and HbA1c levels in Koreans aged ≥50 years.

Relationship between periodontal disease and subclinical atherosclerosis: the Dong-gu study.

AIM: We assessed the association of periodontal disease and number of missing teeth with subclinical atherosclerosis in an adult Korean population. MATERIALS AND METHODS: Cross-sectional data from 5404 individuals aged ≥50 years were obtained from the 2008-2010 Dong-gu study. Periodontal examinations were conducted to determine the number of missing teeth, pocket depth (PD), clinical attachment loss (CAL), and bleeding on probing (BOP). The percentages of sites with PD ≥ 4 mm (PD 4%), CAL ≥ 4 mm (CAL 4%), and BOP (BOP%) were recorded for each participant. B-mode ultrasound was performed to determine common carotid artery intima-media thickness (CCA IMT) and the presence of carotid plaques. Multivariate linear regression models were used to assess the associations between periodontal parameters and CCA IMT and carotid plaque. RESULTS: Number of missing teeth was associated with increased CCA IMT, and BOP% was associated with increased CCA IMT in females only. This association was robust in never smokers. CONCLUSIONS: The number of missing teeth was associated with CCA IMT, and BOP% was associated with CCA IMT in females only. These associations were robust in never smokers. Our results suggest that tooth loss due to oral disease may play a role in subclinical carotid atherosclerosis.

The association between periodontal disease, tooth loss and bone mineral density in a Korean population.

OBJECTIVE: We aimed to evaluate whether clinical attachment loss (CAL), a measure of the severity of periodontal disease or number of teeth present is associated with bone mineral density (BMD). METHODS: The study population consisted of 5383 people aged 50 years and older who participated in the Dong-gu Study. BMD at the lumbar spine and femoral neck was measured by dual-energy X-ray absorptiometry. Oral examination included assessments of the number of teeth present and CAL. Number of teeth present was categorized into three equal categories. CAL values were divided into tertiles in terms of the percentage of sites with CAL ≥4 mm. Analysis of covariance was used to compare the adjusted means of BMD according to the tooth number and the tertiles of CAL. RESULTS: There was a significant association between the number of teeth present and BMD in men. Compared with men with 22 or more teeth, men with 10 and less teeth had lower BMD. CAL was significantly associated with lower BMD at the lumbar spine in women. CONCLUSION: Our data indicate that tooth loss and CAL were associated with low BMD. However, the magnitude of these associations was relatively small and the clinical significance was unclear.

Effects of 625 nm light-emitting diode irradiation on preventing ER stress-induced apoptosis via GSK-3ß phosphorylation in MC3T3-E1.

Prolonged endoplasmic reticulum (ER) stress contributes to cell apoptosis and interferes with bone homeostasis. Although photobiomodulation (PBM) might be used for ER stress-induced diseases, the role of PBM in relieving cell apoptosis remains unknown. During ER stress, glycogen synthase kinase-3ß (GSK-3ß) is critical; however, its functions in PBM remain uncertain. Thus, this study aimed to investigate the role of GSK-3ß in 625 nm light-emitting diode irradiation (LEDI) relieving tunicamycin (TM)-induced apoptosis. Based on the results, pre-625 nm LEDI (Pre-IR) phosphorylated GSK-3ß via ROS production. Compared with the TM group, Pre-IR + TM group reduced the phosphorylation of the α-subunit of eukaryotic translation initiation factor 2 (eIF-2α) and B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax)/Bcl-2 ratio through regulating GSK-3ß. Furthermore, a similar tendency was observed between Pre-IR + TM and Pre-LiCl+TM groups in preventing TM-induced early and late apoptosis. In summary, this study suggests that the Pre-IR treatment in TM-induced ER stress is beneficial for preventing cell apoptosis via GSK-3ß phosphorylation.

Effect of surface treatment on bond strength of Ti-10Ta-10Nb to low-fusing porcelain.

STATEMENT OF PROBLEM: Ti-10Ta-10Nb alloy is a promising alloy for metal ceramic crowns because of its good corrosion resistance and low cytotoxicity. However, more information is needed on the bond strength between this alloy and porcelain. PURPOSE: The purpose of this study was to compare the surface morphology, surface roughness, and bond strength of a Ti-10Ta-10Nb alloy, pure Ti, and a Ti-6Al-4V alloy. MATERIAL AND METHODS: Ti-10Ta-10Nb, pure Ti, and Ti-6Al-4V specimens (25 × 3 × 0.55 mm plate) were prepared and then divided into 6 groups (n=8) according to surface treatment. Group P (control group) was polished with SiC paper. Groups S50 and S250 were airborne-particle abraded with 50 µm and 250 µm aluminum oxide powder. Group HCl was immersed in 10% HCl aqueous solution, and Group HF was immersed in 17% HNO(3)/HF solution. Group TiN was coated with TiN. Atomic force microscopy was used to observe the surface roughness of the metal surface. Scanning electron microscopy was used to analyze the surface profile. A 3-point bending test was performed to evaluate the bond strength. Two-way analysis of variance (ANOVA) was performed to compare the roughness and bond strength and statistical differences were revealed by the Bonferroni post hoc test (α=.05). RESULTS: There were significant differences in the surface roughness, surface profile, and bond strength of the Ti alloys according to the surface treatments. The groups with the higher mean surface roughness showed higher bond strength, but surface profile had a larger effect on the bond strength than surface roughness. Moreover, the bond strength of the Ti-10Ta-10Nb alloy was high. CONCLUSIONS: Ti-10Ta-10Nb would be more suitable for a metal ceramic crown than pure Ti or Ti-6Al-4V, which have limited use because of their low bond strength to porcelain.

Autophagy upregulates inflammatory cytokines in gingival tissue of patients with periodontitis and lipopolysaccharide-stimulated human gingival fibroblasts.

BACKGROUND: Periodontitis is an inflammatory disease caused by multiple disease-associated bacterial species in periodontal tissues. Autophagy is known to modulate various inflammation-driven diseases and inflammatory responses, but the role of autophagy related to the pathogenesis of periodontitis is not fully established. We investigated whether autophagic flux regulated the expression of inflammatory cytokines in the gingiva of periodontitis patients and lipopolysaccharide (LPS)-stimulated human gingival fibroblasts (HGFs) and the underlying mechanism. METHODS: The mRNA and protein expression of proinflammatory cytokines was assessed in human gingival tissues collected from patients with periodontitis and HGFs treated with LPS. The expression of signaling molecules related to autophagy was evaluated by immunofluorescence and Western blot analyses. RESULTS: The expression of interleukin (IL)-6, tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), and intercellular adhesion molecule-1 (ICAM-1) was increased in the gingival tissues of patients with periodontitis. LC3B-positive cells, a typical autophagic marker, were increased in the gingival tissues of periodontitis patients and LPS-treated HGFs. The conversion ratio of LC3-I to LC3-II was higher in the gingival tissues associated with periodontitis and LPS-treated HGFs compared to the controls. The autophagy inhibitor 3-methyladenine (3MA) significantly abrogated the LPS-sustained inflammatory effect by reducing the expression of IL-6, TNF-α, COX-2, and ICAM-1 in HGFs. The phosphorylation of protein kinase B (AKT) and protein S6K1 (S6), signals involved in the mTOR-dependent mechanism, was decreased in gingiva derived from periodontitis patients and LPS-treated HGFs. CONCLUSIONS: Autophagy augmented the production of inflammatory cytokines by mTOR inactivation via the AKT signaling pathway in the gingival tissues of patients with periodontitis and LPS-stimulated HGFs. These findings would provide a better understanding of the mechanism by which autophagy regulates the inflammatory response associated with periodontal pathogenesis.

A Novel Fluorescent Dye Extracted from Buddleja officinalis for Labeling Mitochondria after Fixation.

Mitochondria are versatile organelles and function by communicating with cellular ecosystems. The fluorescent colocalization analysis after fixation is a highly intuitive method to understand the role of mitochondria. However, there are few fluorescent dyes available for mitochondrial staining after fixation. In this study, a novel fluorescent dye (BO-dye), extracted from Buddleja officinalis, was applied for mitochondrial staining in fixed immortalized human oral keratinocytes. The BO-dye (excitation: 414 nm, emission: 677 nm) is a small fluorescent molecular dye, which can cross the cytomembrane without permeabilization. We assume that the BO-dye could aggregate and bind to the mitochondria stably. BO-dye exhibited a mega-Stokes shift (>250 nm), which is an important feature that could reduce self-quenching and enhance the signal-to-noise ratio. Analysis of photophysical properties revealed that the BO-dye is temperature and pH insensitive, and it exhibits superior photostability. These results indicate that BO-dye can be considered an alternative fluorescent dye for labeling mitochondria after fixation.

635 nm LED irradiation may prevent endoplasmic reticulum stress in MC3T3-E1 cells.

Although endoplasmic reticulum (ER) stress is thought to be involved in various diseases such as cancer, metabolic, and inflammatory disorders, the relationship between ER stress and bone diseases, are remains unclear. Tunicamycin-treated MC3T3-E1 osteoblasts were used as the ER stress model in this study. 635 nm light-emitting diode irradiation (635 nm-IR) was carried out for 1 h before and after inducing ER stress. To investigate the effects of 635 nm-IR on ER stress-induced MC3T3-E1 osteoblasts and the underlying mechanism, western blot, reverse transcription polymerase chain reaction, alkaline phosphatase and Alizarin red staining, 2',7'-dichlorodyhydrofluorescein diacetate assay, Fluo-3AM and immunocytochemistry were performed. Pretreatment with 635 nm-IR effectively prevented intracellular reactive oxygen species production and alleviated ER stress through the pancreatic ER kinase (PERK)-eukaryotic initiation factor 2 (eIF2)-activating transcription factor 4 (ATF4)-nuclear factor-like 2 (Nrf2) signaling pathway. Hence, 635 nm-IR may serve a protective role in the treatment of ER stress-related bone diseases.

Isolation of High Purity Mouse Mesenchymal Stem Cells through Depleting Macrophages Using Liposomal Clodronate.

BACKGROUND: The use of mouse bone marrow mesenchymal stem cells (mBMSCs) represents a promising strategy for performing preclinical studies in the field of cell-based regenerative medicine; however, mBMSCs obtained via conventional isolation methods have two drawbacks, i.e., (i) they are heterogeneous due to frequent macrophage contamination, and (ii) they require long-term culturing for expansion. METHODS: In the present study, we report a novel strategy to generate highly pure mBMSCs using liposomal clodronate. This approach is based on the properties of the two cell populations, i.e., BMSCs (to adhere to the plasticware in culture dishes) and macrophages (to phagocytose liposomes). RESULTS: Liposomal clodronate added during the first passage of whole bone marrow culture was selectively engulfed by macrophages in the heterogeneous cell population, resulting in their effective elimination without affecting the MSCs. This method allowed the generation of numerous high-purity Sca-1+CD44+F4/80- mBMSCs (> 95%) with just one passaging. Comparative studies with mBMSCs obtained using conventional methods revealed that the mBMSCs obtained in the present study had remarkably improved experimental utilities, as demonstrated by in vitro multilineage differentiation and in vivo ectopic bone formation assays. CONCLUSION: Our newly developed method, which enables the isolation of mBMSCs using simple and convenient protocol, will aid preclinical studies based on the use of MSCs.

Photochemical reaction to increase melanogenesis using Buddleja officinalis and blue light-emitting diode irradiation in B16F10.

Recently, the incidence of vitiligo has increased because of stresses induced by external environment. Ultraviolet (UV) light therapy is the most commonly used method of treating the disease; however, UV light therapy requires a long treatment period, and prolonged exposure to UV radiation has side effects. The purpose of the present study was to investigate the effects of natural products and LED irradiation (LED-IR) on the synthesis of melanin. It was not possible to effectively increase intracellular melanin production through individual applications of Buddleja officinalis (BO), which is a natural substance selected through screening, or blue light irradiation (Blue-IR). However, when used in combination, these two agents stimulated adenylyl cyclase (AC) and melanin production was induced in the stimulated cells via the CREB/MITF/TYR pathway. Furthermore, the combined treatment with BO and Blue-IR generated low levels of cellular reactive oxygen species (ROS) and induced p38 phosphorylation, which in turn activated MITF in ROS-stimulated synthetic melanocytes, resulting in the promotion of melanogenic pathways other than the CREB/MITF/TYR pathway. In addition, this treatment combination effected melanin transport. These results suggested that the combined therapies can be used to treat melanin-deficiency skin diseases such as vitiligo.