Delivery and Transcriptome Assessment of an In Vitro Three-Dimensional Proximal Tubule Model Established by Human Kidney 2 Cells in Clinical Gelatin Sponges.

The high prevalence of kidney diseases and the low identification rate of drug nephrotoxicity in preclinical studies reinforce the need for representative yet feasible renal models. Although in vitro cell-based models utilizing renal proximal tubules are widely used for kidney research, many proximal tubule cell (PTC) lines have been indicated to be less sensitive to nephrotoxins, mainly due to altered expression of transporters under a two-dimensional culture (2D) environment. Here, we selected HK-2 cells to establish a simplified three-dimensional (3D) model using gelatin sponges as scaffolds. In addition to cell viability and morphology, we conducted a comprehensive transcriptome comparison and correlation analysis of 2D and 3D cultured HK-2 cells to native human PTCs. Our 3D model displayed stable and long-term growth with a tubule-like morphology and demonstrated a more comparable gene expression profile to native human PTCs compared to the 2D model. Many missing or low expressions of major genes involved in PTC transport and metabolic processes were restored, which is crucial for successful nephrotoxicity prediction. Consequently, we established a cost-effective yet more representative model for in vivo PTC studies and presented a comprehensive transcriptome analysis for the systematic characterization of PTC lines.

Sirtuin 6 ameliorates arthritis through modulating cyclic AMP-responsive element binding protein/CCN1/cyclooxygenase 2 pathway in osteoblasts.

INTRODUCTION: CCN1 is an immediate-early gene product pivotal for arthritis progression. We have previously shown that sirtuin 6 (SIRT6) inhibited hypoxia-induced CCN1 expression in osteoblasts. Herein we examined the contribution of cyclic AMP-responsive element binding protein (CREB)/CRE to this suppressive action and the influence of CCN1 on cyclooxygenase (COX) 2 synthesis. MATERIALS AND METHODS: MC3T3-E1 murine osteoblasts were cultured under normoxia (21% oxygen) or hypoxia (2% oxygen). Expressions of CCN1, phospho-CREB (Ser133), COX2 and relevant kinases were assessed by Western blot. SIRT6 was overexpressed in cultured osteoblasts and arthritic joints by a lentiviral-based technique. Activities of CCN1 gene promoter constructs were examined by luciferase reporter assay. Interaction between CREB and CCN1 promoter was assessed by chromatin immunoprecipitation (ChIP). Collagen-induced arthritis (CIA) was established in 20 rats to evaluate the effects of SIRT6 therapy on osteoblastic expressions of phospho-CREB, CCN1 and COX2. RESULTS: SIRT6 suppressed hypoxia-enhanced CCN1 expression and CREB phosphorylation. Attenuation of calcium/calmodulin-dependent protein kinase II (CaMKII) may be responsible for SIRT6-induced CREB inhibition. CRE at - 286 bp upstream of the ATG start codon was essential for CCN1 expression under hypoxia and SIRT6 reduced hypoxia-stimulated CREB/CRE interaction. Forced expression of CREB rescued SIRT6-suppressed CCN1 synthesis. CCN1 induced COX2 expression in osteoblasts. In rat CIA, the therapeutic effect of SIRT6 was accompanied by decreases in osteoblastic expressions of phospho-CREB, CCN1 and COX2. CONCLUSION: Our study indicated that the benefits of SIRT6 to inflammatory arthritis and bone resorption are at least partially derived from its modulation of CREB/CCN1/COX2 pathway in osteoblasts.

Metformin Reduces Bone Resorption in Apical Periodontitis Through Regulation of Osteoblast and Osteoclast Differentiation.

INTRODUCTION: We have previously demonstrated that auxiliary metformin therapy promotes healing of apical periodontitis. Here we aimed to investigate the effects of metformin on osteoblast differentiation and osteoclast formation in cultured cells and rat apical periodontitis. METHODS: Murine pre-osteoblasts MC3T3-E1 and macrophages RAW264.7 were cultured under hypoxia (2% oxygen) or normoxia (21% oxygen) and stimulated with receptor activator of nuclear factor-κB ligand (RANKL) when indicated. Metformin was added to the cultures to evaluate its anti-hypoxic effects. Expressions of osteoblast differentiation regulator runt-related transcription factor 2 (RUNX2), RANKL, and osteoclast marker tartrate-resistant acid phosphatase (TRAP) were assessed by Western blot. Apical periodontitis was induced in mandibular first molars of 10 Sprague-Dawley rats. Root canal therapy with or without metformin supplement was performed. Periapical bone resorption was measured by micro-computed tomography. Immunohistochemistry was used to examine RUNX2, RANKL, and TRAP expressions. RESULTS: Hypoxia suppressed RUNX2 expression and enhanced RANKL synthesis in pre-osteoblasts. TRAP production increased in macrophages after hypoxia and/or RANKL stimulation. Metformin reversed hypoxia-induced RUNX2 suppression and RANKL synthesis in pre-osteoblasts. Metformin also inhibited hypoxia and RANKL-enhanced TRAP synthesis in macrophages. Intracanal metformin diminished bone loss in rat apical periodontitis. Comparing with vehicle control, cells lining bone surfaces in metformin-treated lesions had significantly stronger expression of RUNX2 and decreased synthesis of RANKL and TRAP. CONCLUSIONS: Alleviation of bone resorption by intracanal metformin was associated with enhanced osteoblast differentiation and diminished osteoclast formation in rat apical periodontitis. Our results endorsed the role of metformin as an effective medicament for inflammatory bone diseases.

Isolated Soy Protein Supplementation Combined With Resistance Training Improves Muscle Strength, Mass, and Physical Performance of Aging Female Mice.

Background/Purpose: In recent years, the aging population has gradually increased, and the aging process is accompanied by health-associated problems, such as loss of muscle mass and weakness. Therefore, it is important to explore alternative strategies for improving the health status and physical fitness of the aged population. In this study, we investigated the effect of soy protein supplementation combined with resistance training on changes in the muscle mass, muscle strength, and functional activity performance of aging mice. Methods: Female Institute of Cancer Research (ICR) mice were divided into four groups (n = 8 per group): sedentary control (SC), isolated soy protein (ISP) supplementation, resistance training (RT), and a combination of ISP and RT (ISP + RT). The mice in designated groups received oral ISP supplementation (0.123 g/kg/day), RT (5 days/week for a period of 4 weeks), or a combination of both ISP plus RT for 4 weeks. Afterward, we assessed muscle strength, endurance, and anaerobic endurance performance and analyzed blood biochemical and pathological tissue sections to investigate whether there were adverse effects or not in mice. Results: ISP supplementation effectively improved the muscle mass, muscle endurance, and endurance performance of aging female mice. The RT group not only showed similar results with ISP but also increased muscle strength and glycogen content. Nevertheless, the combination of ISP supplementation and RT had greater beneficial effects on muscle strength, physical performance, and glycogen levels (p < 0.05). In addition, the combination of ISP supplementation and RT had significantly increased type II muscle percentage and cross-sectional area (p < 0.05). Conclusion: Although ISP or RT alone improved muscle mass and performance, the combination of ISP with RT showed greater beneficial effects in aging mice. Our findings suggest that regular exercise along with protein supplementation could be an effective strategy to improve overall health and physical fitness among the elderly.

Differences between the temporal and mandibular components of the temporomandibular joint in topographic distribution of osseous degenerative features on cone-beam computerized tomography.

BACKGROUND/PURPOSE: Temporomandibular joint osteoarthritis (TMJOA) pathology is characterized by degenerative changes of the subchondral bone. The topographic distribution of osseous degenerative changes in TMJ is not clear. This study aimed to evaluate the topographic distribution of osseous degenerative features in the TMJ by using cone-beam computerized tomography (CBCT). MATERIALS AND METHODS: The CBCT images of 26 female patients diagnosed to have TMJOA were retrieved from the database of the National Taiwan University Hospital. The images of left and right TMJs were evaluated independently by 2 examiners. The evaluated degenerative features included surface erosion, subcortical cysts, subcortical sclerosis, and osteophytes in the mandibular condyle and temporal component of the TMJ. The topographic distribution at different portions in the mandibular condyle and temporal component of the TMJ was statistically analyzed. RESULTS: Significant differences in the topographic distribution of the osseous degenerative features were observed (a) between the mandibular condyle and the temporal component and (b) between the anterior/central portion and posterior portion of the temporal component. No significant differences were observed in the topographic distribution of the TMJOA features in the condyle, except for surface erosion between the central and lateral portion of the condyle. CONCLUSION: The results suggest that the mandibular condyle and temporal component react differently in TMJ osseous degeneration, with the condyle being more vulnerable than the temporal component. Mandibular activities that require the mandibular condyle to function outside the fossa may be more destructive to the health and integrity of the TMJ.

The advantage of cone-beam computerized tomography over panoramic radiography and temporomandibular joint quadruple radiography in assessing temporomandibular joint osseous degenerative changes.

BACKGROUND/PURPOSE: The clinical diagnosis of temporomandibular joint (TMJ) degenerative joint disease (DJD) is based primarily on radiographic features of the condyle and articular eminence. The purpose of this study was to compare the reliability, sensitivity, and specificity of using plain radiography to that of cone-beam computerized tomography (CBCT) in identifying different types of osseous degenerative features in the TMJ condyle. MATERIALS AND METHODS: The panoramic radiography (PANO), TMJ quadruple radiography (TMJQR) and CBCT images of 29 patients' TMJs were retrieved from a computer database and independently evaluated by a young oral surgeon and a senior TMD specialist. The examiners diagnosed osseous degenerative features on the radiographic images. The radiologist-assisted CBCT diagnoses were used as a reference standard and the reliability, sensitivity, and specificity of using the three radiographic modalities were statistically analyzed. RESULTS: There were cases of indeterminate diagnoses using the PANO and TMJQR due to superimposition from surrounding structures, but none using CBCT. Reliability was generally poor when using PANO and TMJQR for detecting osseous degenerative features of the TMJ condyle but good to excellent when using CBCT. The sensitivity and specificity in the use of PANO and TMJQR were typically below acceptable, but the levels were generally satisfactory when using CBCT. CONCLUSION: CBCT is superior to plain radiographic modalities for diagnosing osseous degenerative features of TMJs with regard to indeterminate cases, reliability, sensitivity, and specificity. It is recommended that CBCT can be used as an effective tool in identifying TMJ osteoarthritis.

[Expression of EV71-VP1, PSGL-1 and SCARB2 in Tissues of Infants with Brain Stem Encephalitis].

OBJECTIVE: To understand the correlation of enterovirus 71 (EV71), P-selectin glycoprotein ligand-1 (PSGL-1), and scavenger receptor B2 (SCARB2) and to explore the possible pathway and mechanism of EV71 infection by observing the expression of EV71, PSGL-1 and SCARB2 in tissues of infants with brain stem encephalitis. METHODS: The organs and tissues of infants with EV71-VP1 positivity in their brain stems were chosen. Expression and distribution of EV71-VP1, PSGL-1, and SCARB2 were detected and compared by immunohistochemistry. RESULTS: Strong staining of EV71 -VP1 was observed in the neuron, glial cells, the inflammatory cells of perivascular cuffing, parietal cells of the gastric fundus gland while alveolar macrophages, intestinal gland epithelium cells, mucosa lymphoid nodule and lymphocyte of palatine tonsil showed moderate staining and weak staining were displayed in mesenteric lymph nodes and lymphocyte of spleen. PSGL-1 expression was detected in parietal cells of the gastric fundus gland, tonsillar crypt squamous epithelium, alveolar macrophages and leukocytes in each tissue. SCARB2 expression was observed in all the above tissues except the intestines and spleen. CONCLUSION: The distribution of EV71 correlates with SCARB2 expression. SCARB2 plays an important role in virus infection and replication. Stomach may be an important site for EV71 replication.