Spironolactone Attenuates Methylglyoxal-induced Cellular Dysfunction in MC3T3-E1 Osteoblastic Cells

Background@#Methylglyoxal (MG) is associated with the pathogenesis of age- and diabetes-related complications. Spironolactone is a competitive antagonist of aldosterone that is widely employed in the treatment of hypertension and heart failure. This study examined the effects of spironolactone on MG-induced cellular dysfunction in MC3T3-E1 osteoblastic cells. @*Methods@#MC3T3-E1 cells were treated with spironolactone in the presence of MG. The mitochondrial function, bone formation activity, oxidative damage, inflammatory cytokines, glyoxalase I activity, and glutathione (GSH) were measured. @*Results@#Pretreatment of MC3T3-E1 osteoblastic cells with spironolactone prevented MG-induced cell death, and improved bone formation activity. Spironolactone reduced MG-induced endoplasmic reticulum stress, production of intracellular reactive oxygen species, mitochondrial superoxides, cardiolipin peroxidation, and inflammatory cytokines. Pretreatment with spironolactone also increased the level of reduced GSH and the activity of glyoxalase I. MG induced mitochondrial dysfunction, but markers of mitochondrial biogenesis such as mitochondrial membrane potential, adenosine triphosphate, proliferator-activated receptor gamma coactivator 1α, and nitric oxide were significantly improved by treatment of spironolactone. @*Conclusion@#Spironolactone could prevent MG-induced cytotoxicity in MC3T3-E1 osteoblastic cells by reduction of oxidative stress. The oxidative stress reduction was explained by spironolactone's inhibition of advanced glycation end-product formation, restoring mitochondrial dysfunction, and anti-inflammatory effect.

Spironolactone Attenuates Methylglyoxal-induced Cellular Dysfunction in MC3T3-E1 Osteoblastic Cells

Background@#Methylglyoxal (MG) is associated with the pathogenesis of age- and diabetes-related complications. Spironolactone is a competitive antagonist of aldosterone that is widely employed in the treatment of hypertension and heart failure. This study examined the effects of spironolactone on MG-induced cellular dysfunction in MC3T3-E1 osteoblastic cells. @*Methods@#MC3T3-E1 cells were treated with spironolactone in the presence of MG. The mitochondrial function, bone formation activity, oxidative damage, inflammatory cytokines, glyoxalase I activity, and glutathione (GSH) were measured. @*Results@#Pretreatment of MC3T3-E1 osteoblastic cells with spironolactone prevented MG-induced cell death, and improved bone formation activity. Spironolactone reduced MG-induced endoplasmic reticulum stress, production of intracellular reactive oxygen species, mitochondrial superoxides, cardiolipin peroxidation, and inflammatory cytokines. Pretreatment with spironolactone also increased the level of reduced GSH and the activity of glyoxalase I. MG induced mitochondrial dysfunction, but markers of mitochondrial biogenesis such as mitochondrial membrane potential, adenosine triphosphate, proliferator-activated receptor gamma coactivator 1α, and nitric oxide were significantly improved by treatment of spironolactone. @*Conclusion@#Spironolactone could prevent MG-induced cytotoxicity in MC3T3-E1 osteoblastic cells by reduction of oxidative stress. The oxidative stress reduction was explained by spironolactone's inhibition of advanced glycation end-product formation, restoring mitochondrial dysfunction, and anti-inflammatory effect.

Tetrabromobisphenol A Promotes the Osteoclastogenesis of RAW264.7 Cells Induced by Receptor Activator of NF-kappa B Ligand In Vitro

BACKGROUND: Tetrabromobisphenol A (TBBPA), one of the most widely used brominated flame-retardants, is a representative persistent organic pollutants group. Studies on TBBPA toxicity have been conducted using various target cells; however, few studies have investigated TBBPA toxicity in bone cells. Therefore, this study investigated the in vitro effects of TBBPA on osteoclasts, a cell type involved in bone metabolism. METHODS: RAW264.7 cells were cultured in medium containing 50 ng/mL receptor activator of nuclear factor kappa B ligand (RANKL) and varying concentrations of TBBPA. To evaluate the effects of TBBPA on the differentiation and function of osteoclasts, osteoclast-specific gene expression, tartrate-resistant acid phosphatase (TRAP) activity, bone resorbing activity, mitochondrial membrane potential (MMP) and mitochondrial superoxide were measured. RESULTS: The presence of 20 μM TBBPA significantly increased TRAP activity in RANKL-stimulated RAW264.7 cells, the bone resorbing activity of osteoclasts, and the gene expression of Akt2, nuclear factor of activated T-cells cytoplasmic 1, and chloride channel voltage-sensitive 7. However, TBBPA treatment caused no change in the expression of carbonic anhydrase II, cathepsin K, osteopetrosis-associated transmembrane protein 1, Src, extracellular signal-related kinase, GAB2, c-Fos, or matrix metalloproteinase 9. Furthermore, 20 μM TBBPA caused a significant decrease in MMP and a significant increase in mitochondrial superoxide production. CONCLUSION: This study suggests that TBBPA promotes osteoclast differentiation and activity. The mechanism of TBBPA-stimulated osteoclastogenesis might include increased expression of several genes involved in osteoclast differentiation and reactive oxygen species production.

Isolation of Density Enrichment Fraction of Adipose-Derived Stem Cells from Stromal Vascular Fraction by Gradient Centrifugation Method

BACKGROUND: Adipose tissues include multipotent cells, the same as bone marrow-derived mesenchymal stem cells. The stromal vascular fractions (SVFs) from adipose tissues represent a heterogeneous cell population. The purpose of this study was to isolate and purify adipose-derived stem cells (ASCs) in SVFs by the density gradient method. METHODS: SVFs were extracted from the subcutaneous, epididymal, mesenteric and retroperitoneal adipose tissue of 8 weeks old male Sprague-Dawley rats (n = 15) and these were separated into 4 layers according to a Nycodenz gradient (Fx-1: < 11%, Fx-2: 11-13%, Fx-3: 13-19% and Fx-4: 19-30%). The post-confluent SVFs were cultured in adipogenic medium for 2 days, in insulin medium for 2 days and in 10% fetal bovine serum medium for 5 days. To observe lipid droplets in SVFs, we performed Oil Red O staining. RESLTS: The SVFs' cellular fractions (Fx-1, Fx-2, Fx-3 and Fx-4) were isolated by density gradient centrifugation from the adipose tissues of rats. The SVFs extracted to fraction 3 (Fx-3) had the most abundant cells compared to that of the other fractions. However fraction 1 (Fx-1) or 2 (Fx-2) had a superior ability to make lipid droplets. The adipogenic differentiation of Fx-1 or 2 was higher than that of the unfractionated cells. The SVFs extracted from retroperitoneal adipose tissue had the highest efficiency for adipogenic differentiation, whereas the SVFs from mesenteric adipose tissue did not differentiate. CONCLUSION: This density gradient fractionated method leads to efficient isolation and purification of cells with the characteristics of ASCs.

Titanium Mesh Cylinder as an Anterior Strut in Reconstructive Spinal Surgery / 대한척추외과학회지

STUDY DESIGN: A retrospective study. OBJECTIVES: To evaluate the efficiency of titanium mesh cylinder as an anterior strut in reconstructive spinal surgery. SUMMARY OF LITERATURE REVIEW: Biomechanically, 80 to 85% of the axial forces on the upright spine are distributed through the anterior column. Reconstruction of the anterior column using load sharing construct is essential for normal spinal biomechanics in various anterior column deficiency conditions. Titanium mesh cylinder as an anterior strut has advantages in these surgical conditions, and may be an effective alternative to strut bone graft. MATERIALS AND METHODS: From October 1993 to May 1998, 22 patients received reconstructive spinal surgery using titanium mesh cylinder-bone graft composite. Average follow up period was 3(1/2)years(range: 1(1/2)to 6 years). 20 of them were male and 2 were female and average age was 45 years(range: 21 to 69 years). The diagnoses were kyphosis/kyphoscoliosis, burst fracture/ fracture-dislocation, revision surgery, tumor, and multiple thoracic HNP. Radiographs were taken preoperatively, postoper-atively at 2 weeks, 4, 6, 9, 12, 18, 24 months, and yearly thereafter, these were read for change of sagittal angle correction, anterior bony fusion, settling of the titanium mesh cylinder-bone graft composite, or instrument failure. RESULTS: Radiologic union between titanium mesh cylinder & vertebra appeared at (1/2)to 1 (1/2)years follow-up period. 81.8%(18 of 22 cases) showed definite bony trabeculation, and 18.2%(4 of 22 cases) showed immature bony trabeculation. Sagittal angle correction loss was measured by average 7.6 .(range: 5 .to 9 .) in 8 of 22 cases, all of which appeared at 4 months to 1 year follow-up period. Correction loss was associated with penetration of spikes of titanium mesh cylinder into endplates of adjacent vertebral bodies until internal rings contact endplates, but was not accompanied with implant failures, dislodgement or migration of titanium mesh cylinder. CONCLUSIONS: Titanium mesh cylinder can be used as a good substitute of anterior strut in combination with rigid spinal fixa-tion in reconstructive spinal surgery.

Interbody Fusion using Iliac Crest Allograft in Global Spinal Fusion / 대한정형외과학회잡지

PURPOSE: To analyze the efficacy of iliac tricortical allograft (Tutoplast (r) ) as an anterior load sharing construct in global fusion consisting of interbody fusion, transpedicular screw fixation and posterolateral fusion. MATERIALS METHODS: We followed up 40 levels in 27 patients who had undergone interbody fusion using iliac tricortical allograft (Tutoplast (r) ) and added transpedicular screw fixation and poterolateral fusion from Dec. 1995 to Dec. 1996. They were followed up for more than 2 years. The disease entities included 11 patients with spondylolisthesis, 8 patients with spinal stenosis with lumbar intervertebral discs herniation, 6 patients with degenerative lumbar kyphoscoliosis and 2 patients with pseudoarthrosis. Anterior interbody fusion was performed in 11 cases and posterior lumbar interbody fusion in 16 cases. RESULTS: The radiological union rate of interbody fusion was 90% (36/40) , and collapsed union was 7.5% (3/40) ranging from 11% to 28% collapses of initial graft height. The non-union occurred at level 1, which showed radiolucent line on host-graft interface but did not reveal instability on stress views. The radiologic union rate of the posterolateral fusion was 100%. Complications such as metal failure and infection were not noted. Satisfactory clinical results were 88.9%. CONCLUSION: Iliac tricortical allograft was a good substitute for an anterior load sharing construct in interbody fusion combined with transpedicular screw fixation and posterolateral fusion.

Effect of cilostazol on the neuropathies of streptozotocin-induced diabetic rats

OBJECTIVES: This study examined the effect of cilostazol, a potent phosphodiesterase inhibitor, on the progression of neuropathies associated with streptozotocin-induced diabetes mellitus in Sprague-Dawley rats. METHODS: Eight weeks after streptozotocin treatment, a pelleted diet containing 0.03% cilostazol (15 mg/kg body weight) was given for four weeks. Body weight, blood glucose level, motor nerve conduction velocity (MNCV), myelinated fiber density and size distribution of sciatic nerves were compared between age-matched normal rats (Group 1), control diabetic rats (Group 2) and cilostazol-treated diabetic rats (Group 3). RESULTS: Body weight was significantly reduced and blood glucose level was significantly increased in diabetic rats (Group 2 and 3) compared to normal rats. MNCV and cAMP content of sciatic nerves were significantly reduced in diabetic rats 12 weeks after streptozotocin treatment. Myelinated fiber size and density were also significantly reduced, and thickening of the capillary walls and duplication of the basement membranes of the endoneural vessels were observed in the diabetic rats. Whereas both body weight and blood glucose level of Group 3 did not differ significantly from those of Group 2, cilostazol treatment significantly increased MNCV and cAMP content of sciatic nerves in Group 3 but not to the levels observed in Group 1. MNCV positively correlated with cAMP content of sciatic nerves (r = 0.86; p < 0.001). Cilostazol treatment not only restored myelinated fiber density and size distribution but reversed some of the vascular abnormalities. CONCLUSION: These findings suggest that a reduced cAMP content in motor nerves may be involved in the development of diabetic neuropathy, and that cilostazol may prevent the progression of diabetic neuropathy by restoring functional impairment and morphological changes of peripheral nerves.

Effect of glucose on the expression of c-myc gene in cultured RINm5F cell

The study was designed to examine the effect of glucose on the expression of c-myc gene in cultured RINm5F cells. After monolayer culture was established in RPMI 1640 media supplemented with 10% fetal calf serum (FCS), the cells were cultured in various concentrations of glucose and 1 or 10% FCS for another 24 hours. A mRNA was extracted from the cultured cells by a single step method, and Northern analysis was done to detect RNA band. A 0.5 kilobase single band was detected as c-myc mRNA. The expression of c-myc gene mRNA was reduced with increased concentration of glucose with 1% FCS. However, supplementation of 10% FCS abolished the effect of glucose on expression of c-myc gene. These findings suggested that glucose in conjunction with other growth promoting factors played an important role in expression of oncogene and cell growth in RINm5F cells.