Delayed gingival wound healing is widely observed in periodontal patients with diabetes. However, the molecular mechanisms of the impaired function of gingival fibroblasts in diabetes remain unclear. The purpose of this study was to investigate changes in the properties of human gingival fibroblasts (HGFs) under high-glucose conditions. Primary HGFs were isolated from healthy gingiva and cultured with 5.5, 25, 50, and 75 mM glucose for 72 h. In vitro wound healing, 5-ethynyl-2′-deoxyuridine (EdU), and water-soluble tetrazolium salt (WST-8) assays were performed to examine cell migration and proliferation. Lactase dehydrogenase (LDH) levels were measured to determine cytotoxicity. The mRNA expression levels of oxidative stress markers were quantified by real-time PCR. Intracellular reactive oxygen species (ROS) were also measured in live cells. The antioxidant N-acetyl-l-cysteine (NAC, 1 mM) was added to evaluate the involvement of ROS in the glucose effect on HGFs. As a result, the in vitro wound healing assay showed that high glucose levels significantly reduced fibroblast migration and proliferation at 6, 12, 24, 36, and 48 h. The numbers of cells positive for EdU staining were decreased, as was cell viability, at 50 and 75 mM glucose. A significant increase in LDH was proportional to the glucose concentration. The mRNA levels of heme oxygenase-1 and superoxide dismutase-1 and ROS levels were significantly increased in HGFs after 72 h of exposure to 50 mM glucose concentration. The addition of NAC diminished the inhibitory effect of high glucose in the in vitro wound healing assay. The results of the present study show that high glucose impairs the proliferation and migration of HGFs. Fibroblast dysfunction may therefore be caused by high glucose-induced oxidative stress and may explain the delayed gingival wound healing in diabetic patients.

Osteoporosis is one of the most prevalent diseases and the number of patients suffering from this disease is soaring due to the increase in the aged population in the world. The severity of bone loss in osteoporosis is based on the levels of impairment in the balance between bone formation and bone resorption, two arms of the bone metabolism, and bone remodeling. However, determination of bone formation levels is under many layers of control that are as yet fully defined. Bone morphogenetic protein (BMP) plays a key role in regulation of bone formation while its downstream targets are still incompletely understood. Lgr4 gene encodes an orphan receptor and has been identified as a genetic determinant for bone mass in osteoporotic patients. Here, we examine the effects of BMP on the expression of Lgr4 in osteoblastic cells. Lgr4 gene is expressed in an osteoblastic cell line, MC3T3E1 in a time dependent manner during the culture. BMP treatment enhances Lgr4 mRNA expression at least in part via transcriptional event. When Lgr4 mRNA is knocked down, the levels of BMP-induced increase in alkaline phosphatase (Alp) activity and Alp mRNA are suppressed. BMP enhancement of Lgr4 gene expression is suppressed by FGF and reversed by dexamethasone. BMP also enhances Lgr4 expression in primary cultures of calvarial osteoblasts. These data indicate that Lgr4 gene is regulated by BMP and is required for BMP effects on osteoblastic differentiation. J. Cell. Physiol. 231: 887-895, 2016. © 2015 Wiley Periodicals, Inc.

An association between oral diseases and postmenopausal status has been recognized. However, the relationship between all oral disease, mandibular bone density, health status, and osteocalcin (OCN) bone markers in postmenopausal dental patients has not been reported. This study was therefore to verify the differences in plasma OCN levels, dental, periodontal, and oral mucosal disease, and mandibular bone density alterations from panoramic radiograph and systemic parameters in postmenopausal women, compared to premenopausal women. Oral, radiographic, and blood examination were performed in 92 females. Dental, periodontal, and oral mucosal statuses were recorded. Health profile parameters were collected from medical charts. Plasma OCN was evaluated by enzyme-linked immunosorbent assay. Forty-two (45.7%) participants were postmenopausal with a higher median age (55 (51, 62) years) than the premenopausal group (43 (38, 45) years). Overweight or obesity, hypercholesterolemia, and impaired fasting blood sugar were more prevalent in postmenopause. The average postmenopausal OCN level (425.62 ng/mL) was significantly higher than the premenopausal group (234.77 ng/mL, p < 0.001). The average number of missing teeth, mean attachment loss, alveolar bone loss, periapical lesion count, and clinical oral dryness score were also significantly higher in postmenopause (p=0.008,   < 0.001,  0.031,  0.006,  and 0.005, respectively). However, mandibular bone density determined by mandibular cortical index was lower in postmenopause (p < 0.001). The panoramic mandibular index, mandibular cortical width, fractal dimension, and other oral mucosal disease did not differ between the groups. Postmenopause was associated with elevated plasma OCN (β = 0.504, p < 0.001) when related covariates were adjusted. Elevated plasma OCN, oral mucosal dryness, high number of periapical radiolucencies and missing teeth, and lower mandibular bone density from panoramic radiograph were prevalent in postmenopausal women. Dentists should suspect an increased risk of low bone mineral density in postmenopausal patients who display these clinical and radiographic findings, and they should be referred for further examination. Plasma OCN may interconnect a relationship between postmenopausal status and the low mandibular bone density.

LGR4 is expressed in bone and has been shown to be involved in bone metabolism. Oxidative stress is one of the key issues in pathophysiology of osteoporosis. However, the link between Lgr4 and oxidative stress has not been known. Therefore, effects of hydrogen peroxide on Lgr4 expression in osteoblasts were examined. Hydrogen peroxide treatment suppressed the levels of Lgr4 mRNA expression in an osteoblastic cell line, MC3T3-E1. The suppressive effects were not obvious at 0.1 mM, while 1 mM hydrogen peroxide suppressed Lgr4 expression by more than 50%. Hydrogen peroxide treatment suppressed Lgr4 expression within 12 h and this suppression lasted at least up to 48 h. Hydrogen peroxide suppression of Lgr4 expression was still observed in the presence of a transcription inhibitor but was no longer observed in the presence of a protein synthesis inhibitor. Although Lgr4 expression in osteoblasts is enhanced by BMP2 treatment as reported before, hydrogen peroxide treatment suppressed Lgr4 even in the presence of BMP2. Finally, hydrogen peroxide suppressed Lgr4 expression in primary cultures of osteoblasts similarly to MC3T3-E1 cells. These date indicate that hydrogen peroxide suppresses Lgr4 expression in osteoblastic cells. J. Cell. Physiol. 232: 1761-1766, 2017. © 2016 Wiley Periodicals, Inc.