1α,25-Dihydroxyvitamin D3 ameliorates diabetes-induced bone loss by attenuating FoxO1-mediated autophagy.

Autophagy is vital for maintaining cellular homeostasis through removing impaired organelles. It has recently been found to play pivotal roles in diabetes mellitus (DM), which is associated with increased bone fracture risk and loss of bone density. However, the mechanism whereby autophagy modulates DM-induced bone loss is not fully elucidated. Previous work has shown that 1α,25-Dihydroxyvitamin D3 (1,25D) exerts positive effects on autophagy, thus affecting bone metabolism. Here, we investigated whether autophagy was involved in the regulation of diabetic bone metabolism. Using Micro-CT, Elisa, histology, and histomorphometry analysis, we demonstrated that 1,25D rescues glucose metabolism dysfunction and ameliorates bone loss in diabetic mice. In vitro, 1,25D alleviated primary osteoblast dysfunction and intracellular oxidative stress through reducing prolonged high-glucose-mediated excessive autophagy in primary osteoblasts, reflected by decreased protein level of Beclin1 and LC3. Of note, the autophagy activator rapamycin (RAP) ablated the positive effects of 1,25D in diabetic environment, leading to a marked increase in autolysosomes and autophagosomes, examined by mRFP-GFP-LC3 fluorescence double labeling. The excessive autophagy induced by high glucose was deleterious to proliferation and differentiation of primary osteoblasts. Additionally, biochemical studies identified that PI3K/Akt signaling could be activated by 1,25D, resulting in the inhibition of FoxO1. We confirmed that FoxO1 deficiency alleviated high-glucose-induced autophagy and improved biological functions of primary osteoblasts. Together, our results suggest that the PI3K/Akt/FoxO1 signaling pathway is involved in the osteoprotective effect of 1,25D by attenuating autophagy in diabetes, providing a novel insight for the prevention and treatment of diabetes-caused bone loss.

A customized subepithelial connective tissue graft for interdental papilla reconstruction and soft tissue augmentation.

OBJECTIVE: Due to the minimal blood supply and other reasons, stable reconstruction of interdental papilla is difficult. This article presented three cases, which describe a customized subepithelial connective tissue graft aiming to overcome the clinical challenge, with the combination of tunneling technique. CLINICAL CONSIDERATIONS: An original customized subepithelial connective tissue graft combined with tunnel technique aimed to reconstruct interdental papilla (IP). The subepithelial connective tissue graft was partially spilt to create a bowtie-like shape, with four lateral wings and a main body. The four wings were tightly wrapped around the adjacent abutments, and the body part was used to reconstruct the IP. CONCLUSIONS: With the customized subepithelial connective tissue graft, a favorable outcome has been preliminarily confirmed in these cases. Facing patients with a lack of gingival papilla and soft tissue fullness, the customized subepithelial connective tissue graft may be a good choice. CLINICAL SIGNIFICANCE: This study provides a new method to reconstruct IP. The customized subepithelial connective tissue graft may be a good choice when a lack of gingival papilla and soft tissue fullness occurs, which will be of great benefit to meet the aesthetic needs of patients.

1ɑ,25-Dihydroxyvitamin D3 promotes osteogenesis by down-regulating FGF23 in diabetic mice.

1ɑ,25-dihydroxyvitamin D3 (1,25D) and fibroblast growth factor 23 (FGF23) play important roles in bone metabolism through mutual regulation. However, the underlying mechanism between 1,25D and FGF23 in diabetes-induced bone metabolism disorders has not yet been elucidated. In this study, we investigated the effect of 1,25D on FGF23 under diabetic condition both in vitro and in vivo. The results showed that 1,25D down-regulated the expression of FGF23 in osteoblast significantly though a dose-dependent manner in vitro within high glucose environment. Western blot and immunofluorescence analysis indicated that 1,25D activated PI3K/Akt signalling through binding to vitamin D receptor (VDR), which inhibited the phosphorylation of the transcription factor Forkhead Box O1 (FOXO1). Decreased phosphorylation of FOXO1 down-regulated the expression Dickkopf-1 (DKK1), a well-known inhibitor of Wnt signalling. In addition, we observed that 1,25D remarkably ameliorated osteogenic phenotypic markers such as Ocn and Runx2 and rescued diabetes-induced bone loss in vivo. Our results suggested that 1,25D could promote osteogenesis though down-regulating FOXO1/FGF23 in diabetes.

1α,25-Dihydroxyvitamin D3 promotes angiogenesis by alleviating AGEs-induced autophagy.

Diabetes mellitus (DM) induces abnormal angiogenesis and results in multiple chronic vascular complications. Previous studies showed that advanced glycation end products (AGEs) up-regulated in diabetic patients and induced a series of cellular effects such as oxidative stress, inflammation, and autophagy. 1α,25-Dihydroxyvitamin D3 (1,25D), a hormonal form of vitamin D, proved to be beneficial for vascular diseases. However, the underlying mechanism of 1,25D in angiogenesis in DM remains unclear. Using CCK8 assay and transwell assay, we found that 1,25D could partly ameliorate impaired proliferation and migration ability of endothelial cells (ECs) induced by AGEs (200 µg/mL). Furthermore, tube formation assay, Western blot, and real-time qPCR assay were conducted to demonstrate that AGEs impaired angiogenetic ability, and that angiogenesis-related gene expression (i.e., VEGFA, VEGFB, VEGFR1, VEGFR2, TGFß1, MMP2, MMP9) in ECs and 1,25D could promote angiogenesis and angiogenetic markers expression. By using DCFH-DA, ELISA, and Western blot assay, we also found that AGEs-induced oxidative stress impaired angiogenic ability of ECs, and 1,25D alleviated angiogenesis dysfunction by inhibiting oxidative stress. Of note, AGEs-induced excessive autophagy was found to impair angiogenesis. We elucidated that the detrimental autophagy is modulated by 1,25D and AGEs via PI3K/Akt pathway. Observed together, our findings illustrated that AGEs-induced oxidative stress and autophagy resulted in angiogenic disorder and 1,25D improved angiogenesis by restraining excessive autophagy and oxidative stress, providing a novel insight for the treatment of vascular complications in DM.

The role of forkhead box class O1 during implant osseointegration.

FOXO1, a member of the forkhead family of transcription factors, plays a vital role in the osteogenic lineage commitment of mesenchymal stem cells, and affects multiple cellular functions of osteogenic cells. However, prior studies have focused on mesenchymal stem cells but not on differentiated osteoblasts. In addition, studies about the role of FOXO1 during osseointegration are lacking. In this present study, we constructed osteoblast conditional FOXO1 knock-out mice and lentivirus-mediated FoxO1 overexpression to investigate maxillary titanium implant osseointegration. After 4 wk post implant placement, micro-computed tomography, histomorphometric analyses, and RT-qPCR assays were performed. Results showed that compared with the control group, overexpression of FOXO1 significantly enhanced bone formation around implant and bone-implant contact ratio, while loss of FOXO1 impaired peri-implant osteogenesis and osseointegration. Moreover, overexpression of FoxO1 enhanced expression of osteogenesis-related genes, such as Runx2, Alp1, Col1a1, and Bglap. Whereas, knock-out of Foxo1 reduced the expression of osteogenesis-related genes. Taken together, our results suggested that FOXO1 in osteoblasts could enhance osteogenesis-related gene expression to improve osseointegration.

Expression of eosinophil in peripheral blood of patients with COVID-19 and its clinical significance.

AIMS: To investigate the eosinophil cell (EC) expression in peripheral blood of patients infected with severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) and its clinical significance of diagnosis and prognosis. METHODS: 95 patients, whose nucleic acid test of SARS-CoV-2 was positive to make a definite diagnosis of COVID-19, were selected as the study group. They were admitted at the Chengdu Public Health Clinical Medical Center from January 21 to March 2, 2020. Another 95 healthy subjects and 95 non-infectious fever patients during the same period were selected as the control group. The BC-6900 blood cell analyzer was used to continuously observe and detect ECs in 95 patients with COVID-19 and the control group. The differences in expression levels of ECs in peripheral blood were analyzed. RESULTS: ECs were significantly decreased in 95 (75.8%) COVID-19 patients (P < .01). The absolute EC count IQR was 0.01 × 109/L (0 × 109/L - 0.04 × 109/L), and the EC percentage IQR was 0.3% (0.1% - 0.8%). As the patients' condition improved, the ECs returned to normal, but for those without improvement, ECs continued to decline. CONCLUSIONS: ECs decreased remarkably in patients with COVID-19, and gradually returned to normal after the improvement of the patients' condition, while EC continued to decrease in patients without improvement. It is suggested that ECs have certain clinical significance in the diagnosis and prognosis of COVID-19, and may be a useful index in the early warning of acute infectious diseases.

TLR9 activation in large wound induces tissue repair and hair follicle regeneration via γδT cells.

The mechanisms underlying tissue repair in response to damage have been one of main subjects of investigation. Here we leverage the wound-induced hair neogenesis (WIHN) models in adult mice to explore the correlation between degree of damage and the healing process and outcome. The multimodal analysis, in combination with single-cell RNA sequencing help to explore the difference in wounds of gentle and heavy damage degrees, identifying the potential role of toll-like receptor 9 (TLR9) in sensing the injury and regulating the immune reaction by promoting the migration of γδT cells. The TLR9 deficient mice or wounds injected with TLR9 antagonist have greatly impaired healing and lower WIHN levels. Inhibiting the migration of γδT cells or knockout of γδT cells also suppress the wound healing and regeneration, which can't be rescued by TLR9agonist. Finally, the amphiregulin (AREG) is shown as one of most important effectors secreted by γδT cells and keratinocytes both in silicon or in the laboratory, whose expression influences WIHN levels and the expression of stem cell markers. In total, our findings reveal a previously unrecognized role for TLR9 in sensing skin injury and influencing the tissue repair and regeneration by modulation of the migration of γδT cells, and identify the TLR9-γδT cells-areg axis as new potential targets for enhancing tissue regeneration.

Using the intact periosteum for horizontal bone augmentation of peri-implant defects: a retrospective cohort study.

The objectives of this study were to observe hard tissue changes in guided bone regeneration (GBR) with intact periosteum and soft block deproteinised bovine bone mineral (DBBM), and evaluate whether the result of horizontal bone augmentation varied by initial peri-implant defect depth. Forty patients with a single missing tooth and contained peri-implant defect were categorised into three groups according to their presurgical defect depth (≤ 2, 2-4, and 4-6 mm). Cone-beam computed tomography (CBCT) images were collected and reconstructed preoperatively, postoperatively, and at six months' follow up. The buccal bone width (BBW -0, -3, -5), alveolar bone width (ABW -0, -3, -5) and volume of augmented area were measured. At the six-month follow up the increase in BBW, ABW at all levels, and in bone volume, was statistically significant (all p < 0.001). No statistical significance in bone dimensions or bone resorption was found among groups (all p > 0.05). Histological analysis detected new bone formation in intimate contact with bone grafts underlying the periosteum. Within the limitations of this study, the insights gained may be of assistance to suggest that comparable and acceptable results of horizontal bone augmentation can be achieved in cases of peri-implant defect depth of ≤6 mm by means of GBR with intact periosteum.

FOXO1 differentially regulates bone formation in young and aged mice.

It is a great challenge to develop a safe and effective treatment strategy for age-related osteoporosis and fracture healing. As one of the four FOXO transcription factors, FOXO1 is essential for cell proliferation, survival, senescence, energy metabolism, and oxidative stress in various cells. Our previous study demonstrated that specific Foxo1 gene deletion in osteoblasts in young mice results in bone loss while that in aged mice shows the opposite effect. However, the mechanism underlying the differential regulation of bone metabolism by FOXO1 remains to be elucidated. In this study, we generated osteoblast-specific Foxo1 knockout mice by using Foxo1fl/fl and Bglap-Cre mice. In young mice, Foxo1 gene deletion inhibits osteoblast differentiation, leading to a decreased osteoblast number and decreased bone formation rate because of the weakened ability to resist oxidative stress, eventually resulting in bone loss and delayed healing of bone defects. In aged mice, high levels of reactive oxygen species (ROS) promote the diversion of CTNNB1 (ß-catenin) from T cell factor 4 (TCF4)- to FOXO1-mediated transcription, thereby inhibiting Wnt/ß-catenin signaling and leading to decreased osteogenic activity. Conversely, FOXO1 deficiency indirectly promotes the binding of ß-catenin and TCF4 and activates Wnt/ß-catenin signaling, thereby alleviating age-related bone loss and improving bone defect healing. Our study proves that FOXO1 has differential effects on bone metabolism in young and aged mice and elucidates its underlying mechanism. Further, this study provides a new perspective on the treatment of age-related osteoporosis.

Fulminant type 1 diabetes mellitus in pregnancy.

Fulminant type 1 diabetes mellitus (FT1DM) has received clinical attention for its low incidence and poor prognosis. Currently, few cases of FT1DM are associated with pregnancy in clinical practice, but it poses a great threat to the life of mothers and infants. Here, we present two cases of FT1DM in pregnancy. In Case 1, the patient was a 26-year-old woman who was admitted to the hospital with reduced fetal movement. She was diagnosed with FT1DM and delivered a dead female fetus. Lispro and lantus were administered to control blood glucose, and lipoic acid for antioxidant therapy. In Case 2, the patient was a 28-year-old woman who developed nausea, vomiting, diarrhea, and polydipsia, which later proved to be FT1DM. An abortion was induced and blood glucose levels were controlled using an insulin pump. All physicians should be aware of this disease in order to provide prompt diagnosis and emergency treatment, thus improving maternal prognosis. We suggest that plasma glucose/hemoglobin A1C ratio be adopted as a new clinical parameter in predicting FT1DM.

Fulminant type 1 diabetes mellitus in pregnancy

Fulminant type 1 diabetes mellitus (FT1DM) has received clinical attention for its low incidence and poor prognosis. Currently, few cases of FT1DM are associated with pregnancy in clinical practice, but it poses a great threat to the life of mothers and infants. Here, we present two cases of FT1DM in pregnancy. In Case 1, the patient was a 26-year-old woman who was admitted to the hospital with reduced fetal movement. She was diagnosed with FT1DM and delivered a dead female fetus. Lispro and lantus were administered to control blood glucose, and lipoic acid for antioxidant therapy. In Case 2, the patient was a 28-year-old woman who developed nausea, vomiting, diarrhea, and polydipsia, which later proved to be FT1DM. An abortion was induced and blood glucose levels were controlled using an insulin pump. All physicians should be aware of this disease in order to provide prompt diagnosis and emergency treatment, thus improving maternal prognosis. We suggest that plasma glucose/hemoglobin A1C ratio be adopted as a new clinical parameter in predicting FT1DM.