Sirtuin1 promotes osteogenic differentiation through downregulation of peroxisome proliferator-activated receptor γ in MC3T3-E1 cells.

Osteoporosis is a skeletal disorder characterized by bone loss, resulting in architectural deterioration of the skeleton, decreased bone strength and an increased risk of fragility fractures. Strengthening osteogenesis is an effective way to relieve osteoporosis. Sirtuin1 (Sirt1) is a nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase, which is reported to be involved in improving osteogenesis. Sirt1 targets peroxisome proliferator-activated receptor γ (PPARγ) in the regulation of adipose tissues; however, the molecular mechanism of Sirt1 in osteogenic differentiation is still unknown. PPARγ tends to induce more adipogenic differentiation rather than osteogenic differentiation. Hence, we hypothesized that Sirt1 facilitates osteogenic differentiation through downregulation of PPARγ signaling. Mouse pre-osteoblastic MC3T3-E1 cells were cultured under osteogenic medium. Sirt1 was overexpressed through plasmid transfection. The results showed that high expression of Sirt1 was associated with increased osteogenic differentiation, as indicated by quantitative PCR and Western blot analysis of osteogenic markers, and Von Kossa staining. Sirt1 overexpression also directly and negatively regulated the expression of PPARγ and its downstream molecules. Use of the PPARγ agonist Rosiglitazone, reversed the effects of Sirt1 on osteogenic differentiation. Using constructed luciferase plasmids, we demonstrated a role of Sirt1 in inhibiting PPARγ-induced activity and expression of adipocyte-specific genes, including acetyl-coenzyme A carboxylase (Acc) and fatty acid binding protein 4 (Fabp4). The interaction between Sirt1 and PPARγ was further confirmed using co-immunoprecipitation analysis. Together, these results reveal a novel mechanism for Sirt1 in osteogenic differentiation through downregulation of PPARγ activity. These findings suggest that the Sirt1-PPARγ pathway may represent a potential target for enhancement of osteogenesis and treatment of osteoporosis.

miR-218 is involved in the negative regulation of osteoclastogenesis and bone resorption by partial suppression of p38MAPK-c-Fos-NFATc1 signaling: Potential role for osteopenic diseases.

The increased osteoclastic activity accounts for pathological bone loss in diseases including osteoporosis. MicroRNAs are widely accepted to be involved in the regulation of osteopenic diseases. Recently, the low expression of miR-218 was demonstrated in CD14(+) peripheral blood mononuclear cells (PBMCs) from patients with postmenopausal osteoporosis. However, its role and the underlying mechanism in osteoporosis are still undefined. Here, an obvious decrease in miR-218 expression was observed during osteoclastogenesis under receptor activator of nuclear factor κB ligand (RANKL) stimulation, in both osteoclast precursors of bone marrow macrophages (BMMs) and RAW 264.7. Further analysis confirmed that overexpression of miR-218 obviously attenuated the formation of multinuclear mature osteoclasts, concomitant with the decrease in Trap and Cathepsin K levels, both the master regulators of osteoclastogenesis. Moreover, miR-218 up-regulation dramatically inhibited osteoclast precursor migration, actin ring formation and bone resorption. Mechanism assay demonstrated that miR-218 overexpression attenuated the expression of p38MAPK, c-Fos and NFATc1 signaling molecules. Following preconditioning with P79350, an agonist of p38MAPK, the inhibitor effect of miR-218 on osteoclastogenesis and bone-resorbing activity was strikingly ameliorated. Together, this study revealed a crucial role of miR-218 as a negative regulator for osteoclastogenesis and bone resorption by suppressing the p38MAPK-c-Fos-NFATc1 pathway. Accordingly, this research will provide a promising therapeutic agent against osteopenic diseases including osteoporosis.

Inhibitors of Growth 1b Suppresses Peroxisome Proliferator-Activated Receptor-ß/δ Expression Through Downregulation of Hypoxia-Inducible Factor 1α in Osteoblast Differentiation.

Bone formation, a highly regulated developmental process, involves osteoblast differentiation, which is controlled by different important transcription factors. Recent evidence has suggested possible negative regulation of inhibitors of growth (ING) 1b on the osteoblast marker expression. The aim of this study is to examine the detailed mechanism by which the activity of ING1b inhibits osteoblast differentiation. In the current study, we investigated the function and mechanism by which ING1b inhibits osteoblast differentiation using C3H10T1/2 mesenchymal stem cells and MC3T3-E1 preosteoblasts. Real-time polymerase chain reaction and Western blotting showed that ING1b was decreased during osteoblast differentiation and ING1b overexpression markedly decreased alkaline phosphatase (ALP) activity, runt-related transcription factor 2 (Runx2) expression, and collagen type 1 synthesis, whereas ING1b silencing significantly upregulated ALP activity, Runx2 expression, and collagen type 1 synthesis. Further studies indicated that ING1b suppressed the expression of peroxisome proliferator-activated receptor (PPAR)-ß/δ in a hypoxia-inducible factor (HIF) 1α-dependent manner, while ING1b silencing significantly increased the expression of PPAR-ß/δ and HIF1α. Moreover, PPAR-ß/δ or HIF1α silencing significantly inhibited ALP activity, Runx2 expression, and collagen type 1 synthesis. These results demonstrated that ING1b is an important regulator of osteoblast differentiation and suppresses PPAR-ß/δ. Our study may provide additional insight into osteoblast differentiation and offer a potential new molecular target for osteoporosis.

Single level anterior interbody fusion and fixation in the treatment of thoracolumbar fractures.

BACKGROUND: Many surgical methods are available for repairing thoracolumbar fractures including short-segment internal fixation with posterior pedicle screws and anterior decompression and reduction. However, most methods are associated with significant surgical trauma and long postoperative recovery. The purpose of this study was to describe anterior single level interbody fusion and fixation for the repair of thoracolumbar fractures which may reduce surgical trauma and help speed recovery. METHODS: A group of 21 patients who underwent single level anterior interbody fusion and fixation from June 2006 to June 2011 were compared with a group of 21 patients who underwent double level anterior interbody fusion and fixation during the same period. The groups were compared with regard to operation time, intraoperative blood loss, fracture healing time, ratio of pre- to postoperative endplate height between adjacent vertebrae, Cobb angle in the sagittal plane, recovery of neural function, and internal fusion failure. RESULTS: The 2 groups were similar with the exception of fracture location (P=0.017). The patients who underwent the single level procedure had a shorter operation time (P < 0.001), less blood loss (P < 0.001), and shorter follow-up (P < 0.001). Both groups had significant improvement in Cobb angle at 1 week and 1 year after surgery, but there was no significant difference between the groups. Both groups also exhibited improvement in neurological function, and the difference in improvement between the groups was not significant. CONCLUSIONS: Single level intervertebral fusion and internal fixation for thoracolumbar fractures provides as satisfactory an outcome as the traditional approach, double level anterior interbody fusion and fixation, and reduces the degree of surgical trauma.

[Study on evaluating sex determining region of the Y as an engrafting track of BMSCs transplantation for repairing osteonecrosis of the femoral head of rabbit].

OBJECTIVE: To evaluate sex determining region of the Y (Sry) as a engrafting track of the transplanted BMSCs survival and new bone formation in the osteonecrosis of the femoral head (ONFH) of rabbit. METHODS: Forty-nine 4-5-month-old New Zealand White rabbits were included, weighing 2.0-2.5 kg, 48 females and 1 male. BMSCs of the rabbits were isolated by density gradient separation method, the third passage cells were marked by 1, 1'-dioctadecyl-3, 3, 3', 3'-tetramethylindocarbocyanine perchlorate (DiI) and the concentration of cell suspension was 2.5 x 10(8)/mL. The animal model of ONFH were established with 48 female rabbits by injecting liquid nitrogen, and femoral head was not dislocated. The animal model were divided into 3 groups, 16 rabbits in each group. Group A only established animal model as control. Autologous BMSCs (4 microL) marked by DiI was transplanted in the ONFH models of the group B. Allogenic BMSCs (4 microL) marked by DiI was transplanted in ONFH models of the group C. The femoral head were observed by X-ray, HE staining and Masson staining, and the regenerating trabecular volume percentages was determined at 2, 4, 6 and 8 weeks after operation respectively. The examples of the heart, lung, liver, spleen and kidney were obtained. The transplanted BMSCs were traced by fluorescence microscope, the Sry gene expression was detected by PCR for cells survival. RESULTS: All rabbits survived till the end of experiment. The X-ray showed gradual necrosis in the femoral head of group A. HE and Masson staining results indicated that compared with the group A, the recovery condition of the necrotic femoral head in the groups B and C was better. At each time of groups B and C, the regenerating trabecular volume percentages were higher than that of the group A significantly (P < 0.01). There was no significant difference between groups B and C (P > 0.05). The cells marked by DiI were not founded in the tissues of the heart, lung, liver, spleen and kidney in groups B and C at each time. PCR showed that the expression of Sry gene were not observed at the heart, lung, liver, spleen and kidney of three groups at each time. The expression of Sry gene was clearly identified in the femoral head of all 16 rabbits in the group C at each time point. CONCLUSION: Allografting of BMSCs transplanted into the femoral head can survive and induce new bone formation without redistribution.

[Features of crush injury in Wenchuan earthquake and the corresponding operational methods].

OBJECTIVE: To investigate the characteristics of patients with crush injury in Wenchuan earthquake and the corresponding operational methods. METHODS: From May 12th 2008 to June 18th 2008, 202 patients with crush injury of soft tissue were treated, including 110 males and 92 females. Twenty-five patients aged 19 months to 16 years, 129 patients aged 17-60 years and 48 patients aged above 61 years. The crushed time was 30 minutes to 154 hours. Sixty cases of open injuries were treated by debridement and dressing or suture; 16 cases of damaged extremities (18 limbs) and 6 cases of acute renal failure due to crush syndrome (8 limbs) received amputation; 32 cases of interfascial space syndrome crisis (42 limbs) were treated by fascia cavity decompression; 15 cases received the resection of necrotic muscle for 31 times; and 9 cases received continuous renal replacement therapy (CRRT). RESULTS: All the wounds healed except 2 cases which died from intestinal bleeding and intracranial hemorrhage during the treatment of CRRT. Two cases were discharged 8 months after treatment, while the other 198 cases recovered and were discharged 15-120 days after treatment. The average hospitalization time was 53 days. Twenty-two cases (26 limbs) were fixed with artificial limbs 3-6 months after amputation and achieved good functional outcome. CONCLUSION: The treatment principle of crush injury is "be active to decompress and be prudent to amputate", the hardening muscle and the increasing level of creatine kinase and blood potassium are the golden indicators of fascia cavity decompression. Decompression at an earlier period is preferred when there is a dilemma to choose, and open amputation should be performed when the necrotic muscle is hard to clear or the necrosis boundary is not distinct.

[The clinical effect of anti-rotation reduction internal fixator on the treatment of fresh thoracolumbar spine fracture].

OBJECTIVE: To evaluate the effect of self-designed anti-rotation reduction internal fixator (ARRIF) on treating different spine segment fracture. METHODS: From August 1999 to March 2003, 76 patients(48 males and 28 females, aged from 22 to 59 with an average of 34.1) with thoracolumbar fracture were operatively treated by ARRIF. The follow-up period ranged from 6 to 21 months (15 months in average). Classification according to injury segment: flexion compression fracture 27 cases, burst fracture 42 cases, flexion distraction injury 3 cases, flexion revolving type fracture dislocation 2 cases, shear force type dislocation 2 cases. Classification according Frankel's grade: A grade 16 cases, B grade 15 cases, C grade 27 cases, D grade 10 cases, E grade 8 cases. Operation duration, volume of bleeding, incidence post-operation complication and effect of reduction-fixation were observed. RESULTS: The operation duration of ARRIF was 1.2 h in average, and there was about 200 ml volume of bleeding during operation. The nerve function showed one Frankel's grade improvement after operation were as follows: A grade 8 cases (50%), B grade 11 cases (73.3%), C grade 20 cases (74.1%), D grade 3 cases (30%); 2 Frankel's E cases have no nerve function changes. The nerve function damage have no aggravation in all the patients, the postoperation Cobb's angle was averagely corrected 22 degrees. The horizontal displacement of dislocation vertebrae was averagely corrected 28% in sagittal plane, the statistical analysis had significant variance (P < 0.01). ARRIF had no complications of the breakage of screws and rods. CONCLUSION: ARRIF proves to be a valid internal fixator in reducing and fixing different thoracic lumbar segment spine fracture.