Ginsenoside Rb2 exhibits therapeutic value for male osteoporosis in orchiectomy mice by suppressing osteoclastogenesis and modulating NF-κB/MAPK signaling pathways.

Osteoporosis (OP) is a systemic disorder characterized by decreased bone mass as well as deteriorated microarchitecture. Although OP in men is common, it has received much less attention than that in women. Ginseng, a famous traditional herb in Asia, is used to strengthen and repair bones by invigorating vital bioenergy and maintaining body homeostasis in dietary intake and clinical applications. However, there is currently no study investigating the impact of ginseng and its active compounds on male osteoporosis. In this study, RNA sequencing and bioinformatic analysis were conducted to reveal the influence of Ginsenoside-Rb2 on RAW264.7 cells and its underlying signaling pathways. The potential anti-osteoporosis effects of Rb2 as well as its molecular mechanisms were elucidated in RAW264.7 cells and BMMs by TRAP staining, F-actin belt staining, qRT-PCR and WB. Moreover, orchiectomy (ORX) was utilized to demonstrate the influence of Rb2 on bone mass loss in vivo by micro-CT scanning, and H&E, TRAP, and IHC staining. The results suggested that Rb2 suppressed osteoclastogenesis and mitigated bone loss in orchiectomy mice through NF-κB/MAPK signaling pathways. These findings indicate that ginseng as well as its active component Rb2 have potential therapeutic value in the management of osteoporosis in men.

Myricitrin promotes osteogenesis and prevents ovariectomy bone mass loss via the PI3K/AKT signalling pathway.

This study aimed to explore the effect of myricitrin on osteoblast differentiation in mice immortalised bone marrow mesenchymal stem cells (imBMSCs). Additionally, ovariectomy (OVX) mice were employed to examine the effect of myricitrin on bone trabecular loss in vivo. The effect of myricitrin on the proliferation of imBMSCs was evaluated using a cell counting kit-8 assay. Alizarin red staining, alkaline phosphatase staining were performed to elucidate osteogenesis. Furthermore, qRT-PCR and western blot determined the expression of osteo-specific genes and proteins. To screen for candidate targets, mRNA transcriptome genes were sequenced using bioinformatics analyses. Western blot and molecular docking analysis were used to examine target signalling markers. Moreover, rescue experiments were used to confirm the effect of myricitrin on the osteogenic differentiation of imBMSCs. OVX mice were also used to estimate the delay capability of myricitrin on bone trabecular loss in vivo using western blot, micro-CT, tartaric acid phosphatase (Trap) staining, haematoxylin and eosin staining, Masson staining and immunochemistry. In vitro, myricitrin significantly enhanced osteo-specific genes and protein expression and calcium deposition. Moreover, mRNA transcriptome gene sequencing and molecular docking analysis revealed that this enhancement was accompanied by an upregulation of the PI3K/AKT signalling pathway. Furthermore, copanlisib, a PI3K inhibitor, partially reversed the osteogenesis promotion induced by myricitrin. In vivo, western blot, micro-CT, hematoxylin and eosin staining, Masson staining, Trap staining and immunochemistry revealed that bone trabecular loss rate was significantly alleviated in the myricitrin low- and high-dose groups, with an increased expression of osteopontin, osteoprotegerin, p-PI3K and p-AKT compared to the OVX group. Myricitrin enhances imBMSC osteoblast differentiation and attenuate bone mass loss partly through the upregulation of the PI3K/AKT signalling pathway. Thus, myricitrin has therapeutic potential as an antiosteoporosis drug.

Quercetin protects against iron overload-induced osteoporosis through activating the Nrf2/HO-1 pathway.

AIMS: Eucommia is the tree bark of Eucommia japonica, family Eucommiaceae. In traditional Chinese medicine, Eucommia is often used to treat osteoporosis. Quercetin (QUE), a major flavonoid extract of Eucommia japonica, has been reported to have anti-osteoporosis effects. However, there are no studies reporting the mechanism of QUE in the treatment of iron overload-induced osteoporosis. This study set out to investigate the therapeutic effects of QUE against iron overload-induced bone loss and its potential molecular mechanisms. MATERIALS AND METHODS: In vitro, MC3T3-E1 cells were used to study the effects of QUE on osteogenic differentiation, anti-apoptosis and anti-oxidative stress damage in an iron overload environment (FAC 200 µM). In vivo, we constructed an iron overload mouse model by injecting iron dextrose intraperitoneally and assessed the osteoprotective effects of QUE by Micro-CT and histological analysis. KEY FINDINGS: In vitro, we found that QUE increased the ALP activity of MC3T3-E1 cells in iron overload environment, promoted the formation of bone mineralized nodules and upregulated the expression of Runx2 and Osterix. In addition, QUE was able to reduce FAC-induced apoptosis and ROS production, down-regulated the expression of Caspase3 and Bax, and up-regulated the expression of Bcl-2. In further studies, we found that QUE activated the Nrf2/HO-1 signaling pathway and attenuated FAC-induced oxidative stress damage. The results of the in vivo study showed that QUE was able to reduce iron deposition induced by iron dextrose and attenuate bone loss. SIGNIFICANCE: Our results suggested that QUE protects against iron overload-induced osteoporosis by activating the Nrf2/HO-1 signaling pathway.

Comparison of pedicle screw fixation with or without cement augmentation for treating single-segment isthmic spondylolisthesis in the osteoporotic spine.

The present study examined the necessity of cement-augmented pedicle screw fixation in osteoporotic patients with single-segment isthmic spondylolisthesis.Fifty-nine cases were reviewed retrospectively. Thirty-three cases were in the polymethylmethacrylate-augmented pedicle screw (PMMA-PS) group, and the other 26 cases were in the conventional pedicle screw (CPS) group. Evaluation data included operation time, intraoperative blood loss, hospitalization cost, hospitalization days, rates of fusion, screw loosening, bone cement leakage, visual analogue scale (VAS) scores, Oswestry disability index (ODI), lumbar lordosis (LL), pelvic tilt (PT) and sacral slope (SS).The operation time and blood loss in the CPS group decreased significantly compared to those in the PMMA-PS group. The average hospitalization cost of the PMMA-PS group was significantly higher than that of the CPS group. There was no significant difference in the average hospital stay between the 2 groups. The initial and last follow-up postoperative VAS and ODI scores improved significantly in the two groups. There were no significant differences in VAS and ODI between the 2 groups at each time point. The last postoperative spine-pelvic parameters were significantly improved compared with those preoperatively. In the PMMA-PS group, the fusion rate was 100%. The fusion rate was 96.15% in the CPS group. No significant difference was found between the two groups for the fusion rate. Nine patients in the PMMA-PS group had bone cement leakage. There was no screw loosening in the PMMA-PS group. There were 2 cases of screw loosening in the CPS group. There were no significant differences in screw loosening, postoperative adjacent segment fractures, postoperative infection or postoperative revision between the 2 groups. The use of PMMA-PS on a regular basis is not recommended in posterior lumbar interbody fusion for the treatment of single-segment isthmic spondylolisthesis with osteoporosis.

Bioinformatics analysis combined with experimental validation to explore the mechanism of XianLing GuBao capsule against osteoarthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: XianLing GuBao Capsule (XLGB) is often used to treat osteoarthritis (OA), osteoporosis, fractures, and other musculoskeleton disorders. However, the molecular mechanism of XLGB for treating OA is still unclear. AIM OF THE STUDY: This study set out to uncover the molecular mechanism underlying the treatment of osteoarthritis with XLGB. MATERIALS AND METHODS: Disease genes were obtained from CTD, DisGeNET, and GeneCards databases, and XLGB drug targets were obtained from ETCM and target genes predicted by XLGB metabolic components reported in the literature. Then we used the Venn diagram viewer to extract disease and drug intersection genes as potential therapeutic genes for Protein-protein interaction (PPI), GO terminology, and KEGG pathway analysis. Subsequently, we performed qRT-PCR, Western blot and histological analysis to validate the therapeutic effect of XLGB against OA and its molecular mechanism. RESULTS: A total of 1039 OA genes and 949 XLGB target genes were collected, and finally 188 potential therapeutic target genes were obtained. PPI network analysis indicated that the main target genes for XLGB to treat OA include Akt1, Mapk3, Il-6, Il-1ß, Ptgs2, Mmp9, etc. The results of KEGG and GO enrichment analysis suggested that XLGB may treat OA by anti-inflammatory and reducing extracellular matrix degradation. In vitro, XLGB down-regulated the expressions of Mmp3, Mmp9, Mmp12, Mmp13, Cox-2, Il-6, increased the expression of Collagen II and Sox9. Mechanistically, XLGB inhibits the activation of PI3K/AKT/NF-κB and MAPK pathways. Moreover, the results of animal experiments indicated that XLGB reduced cartilage destruction, bone resorption, and synovitis in osteoarthritic rats. CONCLUSIONS: XLGB has a protective effect against OA by suppressing PI3K/AKT/NF-κB and MAPK signaling. Our study provides a theoretical basis for XLGB in the treatment of osteoarthritis.

Long-Term Outcomes of Peripheral Pulmonary Cement Embolism in Patients with Polymethylmethacrylate Augmentation: A Case Series with a Minimum Follow-Up of Five Years.

BACKGROUND: Pulmonary cement embolism (PCE) is a rare but lethal complication. However, few long-term follow-up studies have investigated PCE after polymethylmethacrylate augmentation. This study aimed to investigate both the clinical and imaging outcomes of patients with PCE during a follow-up period of at least 5 years. METHODS: A total of 1460 patients were initially included in this retrospective study. After exclusion, the clinical and imaging data were analyzed for selected patients, including the augmented level, location and length of the PCE, symptoms, therapy, migration and disintegration of the embolism, foreign body reaction, and status at follow-up. RESULTS: Twelve female patients (age range, 56-88 years) with PCE and more than 5 years of follow-up (range, 5-13 years) were eventually included. All emboli were found in subsegment pulmonary arteries and were classified as peripheral PCE. Although 2 patients experienced transient symptoms after surgery, the majority of patients (84.6%) were asymptomatic during follow-up. No other reported emboli were observed during the follow-up period. The imaging data showed that the cement embolus could remain in the initial position throughout the long-term follow-up. In terms of the length of the PCE, there was no statistically significant difference between the values post-operation and at the last follow-up time (P > 0.05). CONCLUSIONS: Patients with peripheral PCE do not develop known late complications. Moreover, polymethylmethacrylate can remain stable and inert in the pulmonary vasculature over the long term. Routine prophylactic anticoagulation may not be necessary for patients with peripheral PCE during follow-up.

Carnosol suppresses RANKL-induced osteoclastogenesis and attenuates titanium particles-induced osteolysis.

Osteolysis is a common medical condition characterized by excessive activity of osteoclasts and bone resorption, leading to severe poor quality of life. It is essential to identify the medications that can effectively suppress the excessive differentiation and function of osteoclasts to prevent and reduce the osteolytic conditions. It has been reported that Carnosol (Car), isolated from rosemary and salvia, has anti-inflammatory, antioxidative, and anticancer effects, but its activity on osteolysis has not been determined. In this study, we found that Car has a strong inhibitory effect on the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation dose-dependently without any observable cytotoxicity. Moreover, Car can inhibit the RANKL-induced osteoclastogenesis and resorptive function via suppressing NFATc1, which is a result of affecting MAPK, NF-κB and Ca2+ signaling pathways. Moreover, the particle-induced osteolysis mouse model confirmed that Car could be effective for the treatment of bone loss in vivo. Taken together, by suppressing the formation and function of RANKL-induced osteoclast, Car, may be a therapeutic supplementary in the prevention or the treatment of osteolysis.

Influence of cement-augmented pedicle screws with different volumes of polymethylmethacrylate in osteoporotic lumbar vertebrae over the adjacent segments: a 3D finite element analysis.

BACKGROUND: Polymethylmethacrylate (PMMA) is commonly used for cement-augmented pedicle screw instrumentation (CAPSI) to improve the fixation stability and reduce the risk of screw loosening in the osteoporotic thoracolumbar spine. Biomechanical researches have shown that various dose of cement (1-3 ml) can be injected to enhance screw stability. To date, there have been no studies on the relationship between adjacent segment degeneration and the volume of PMMA. This study aimed to explore the influence of CAPSI with different volumes of PMMA in osteoporotic lumbar vertebrae over adjacent segments by using finite element analysis. METHODS: Seven different finite element models were reconstructed and simulated under different loading conditions, including (1) an intact model, (2) three single-level CAPSI models with different volumes of PMMA (1, 1.73, and 2.5 ml), and (3) three double-level CAPSI models with different volumes of PMMA (1, 1.73, and 2.5 ml). To improve the accuracy of the finite element analysis, the models of the injectable pedicle screw and bone cement were created by using a three-dimensional scanning machine and the CAPSI patient's CT data, respectively. The range of motion (ROM), the stress of intervertebral discs, and the stress of facet in the adjacent segment were comparatively analyzed among the different models. RESULTS: The ROMs of the different segments were compared with experimental data, with good agreement under the different load conditions (21.3°, 13.55°, 13.99°, and 6.11° in flexion, extension, bending, and rotation at L3-S1 level, respectively). Compared with the intact model, the ROM, disc stresses, and facet stress in adjacent segments were found to be higher in the six operative models. Otherwise, with a larger volume of PMMA injected, the ROM, disc stresses, and facet stress slightly increased at the adjacent segment. However, the differences were insignificant with the biggest difference less than 3.8%. CONCLUSIONS: CAPSI could increase the incidence of disk degeneration in the adjacent segment, while within a certain range, different volumes of PMMA provided an approximate impact over the adjacent segment degeneration.

Pedicle Screw Fixation in Single-Level, Double-Level, or Multilevel Posterior Lumbar Fusion for Osteoporotic Spine: A Retrospective Study with a Minimum 2-Year Follow-Up.

BACKGROUND: Pedicle screw loosening is a common postoperative complication for osteoporotic patients, and several studies have identified the important role of fusion length in internal fixation failure, but the relationship between the number of fusion segments and the potential risks remains unclear. This study aimed to investigate the rate and risk factors of screw loosening in osteoporotic patients with different levels of degenerative lumbar disease. METHODS: The total cohort of 217 patients was divided into 3 groups according to the different fusion levels: single-level (group A; 100 cases), double-level (group B; 73 cases), and multilevel group (group C; 44 cases). Patient baseline demographic characteristics and assessments with a visual analog scale (VAS) and the Oswestry Disability Index (ODI) before operation and at the last follow-up were compared among the 3 groups. RESULTS: Compared with preoperative values, VAS and ODI scores at the last follow-up were significantly improved in all 3 groups. Operative time, blood loss, length of hospital stay, screw loosening rate, fusion rate, and VAS and ODI scores at the last follow-up obviously increased with the increasing number of fusion segments (group C> group B> group A). Of note, all the screw loosening was observed in cranial and caudal vertebra. Furthermore, multivariate logistic regression analysis identified lumbosacral fixation, larger pelvic incidence (PI)-lumbar lordosis (LL) difference (PT-LL), and greater postoperative pelvic tilt (PT) as independent predictors of screw loosening. However, sex, bone mineral density, body mass index, LL, sacral slope, PI, the change in LL, and preoperative PT were not relevant to screw loosening (P > 0.05). CONCLUSIONS: Owing to the high rate of screw loosening in cranial and caudal vertebra, osteoporotic patients with double-level or multilevel pedicle screw fixation benefited less than those with single-level pedicle screw fixation. Larger PI-LL, larger PT, and lumbosacral fixation are other risk factors for screw loosening. An instrument with stronger holding strength at cranial and caudal pedicle screws is recommended for those high-risk patients.

Effect and potential risks of using multilevel cement-augmented pedicle screw fixation in osteoporotic spine with lumbar degenerative disease.

BACKGROUND: The increase of augmented level and bone cement dose are accompanied by the rising incidence of cement leakage (CL) of cement-augmented pedicle screw instrumentation (CAPSI). But the effect and potential risks of the application of CAPSI to osteoporotic lumbar degenerative disease (LDD) have not been studied in the case of multilevel fixation. This study aimed to investigate the effectiveness and potential complications of using multilevel CAPSI for patients with osteoporotic LDD. METHODS: A total of 93 patients with multilevel LDD were divided into the CAPSI group (46 subjects) and the conventional pedicle screw (CPS) group (47 subjects), including 75 cases for three levels and 18 cases for four levels. Relevant data were compared between two groups, including baseline data, clinical results, and complications. RESULTS: In the CAPSI group, a total of 336 augmented screws was placed bilaterally. The CL was observed in 116 screws (34.52%). Three cemented screws (0.89%) were found loosened during the follow-up and the overall fusion rate was 93.47%. For perioperative complications, two patients (4.35%) experienced pulmonary cement embolism (PCE), one patient augmented vertebral fracture, and three patients (6.52%) wound infection. And in the CPS group, thirty-three screws (8.46%) suffered loosening in cranial and caudal vertebra with a fusion rate of 91.49%. The operation time and hospital stay of CAPSI group were longer than the CPS group, but CAPSI group has a lower screw loosening percentage (P<0. 05). And in terms of blood loss, perioperative complications, fusion rate, and VAS and ODI scores at the follow-up times, there were no significant differences between the two groups. CONCLUSIONS: Patients with osteoporotic LDD underwent multilevel CPS fixation have a higher rate of screw loosening in the cranial and caudal vertebra. The application of cemented pedicle screws for multilevel LDD can achieve better stability and less screw loosening, but it also accompanied by longer operating time, higher incidence of CL, PCE and wound infections. Selective cement augmentation of cranial and caudal pedicle screws may be a worthy strategy to decrease the complications.

Stability Evaluation of Oblique Lumbar Interbody Fusion Constructs with Various Fixation Options: A Finite Element Analysis Based on Three-Dimensional Scanning Models.

BACKGROUND: Little is known about the biomechanical performance of various fixation constructs after oblique lumbar interbody fusion (OLIF). This study aimed to explore the stability of various fixation options for OLIF by using finite element analysis based on three-dimensional scanning models. METHODS: Six validated finite element models of the L3-L5 segment were reconstructed via computed tomography images, including (1) intact model, (2) stand-alone model with no instrument, (3) lateral rod-screw model, (4) lateral rod-screw plus contralateral translaminar facet screw (LRS-CTLFS) model, (5) unilateral pedicle screw model, and (6) bilateral pedicle screw (BPS) model. Models of the OLIF cage and pedicle screw were created with three-dimensional scanning to improve the accuracy of finite element analysis. Range of motion, stress of the cage, and stress of fixation were evaluated in the different models. RESULTS: Range of motion increased from least to greatest as follows: BPS, LRS-CTLFS, unilateral pedicle screw, lateral rod-screw, stand-alone. Differences in range of motion between BPS and LRS-CTLFS were not significant for all loading cases. Compared with the other 3 models, the stress of the cage was found to be lower in BPS and LRS-CTLFS under all loading conditions, especially in BPS. Stress exerted on the fixation was the greatest in LRS-CTLFS, and the stress experienced by the translaminar facet screw was concentrated in part of the facet joint. CONCLUSIONS: The BPS model provided the best biomechanical stability for OLIF; the stand-alone model could not provide sufficient stability. The LRS-CTLFS procedure increases the approximate stability and reduces stress at the cage-endplate interface; however, it causes an increase in screw stress.

Risk Factor Analysis of the Incidence of Subsequent Adjacent Vertebral Fracture After Lumbar Spinal Fusion Surgery with Instrumentation.

OBJECTIVE: This study aimed to evaluate the risk factors for adjacent vertebral compression fractures after lumbar spinal fusion with instrumentation. METHODS: A total of 669 patients who received lumbar instrumented spinal fusion between January 2012 and December 2015 were divided into 2 groups according to whether the adjacent vertebral body was fractured. The covariates recorded were age, sex, bone mineral density, and the number of fixed segments. The anatomic variables were pelvic incidence angle (PI), preoperative lumbar lordosis angle (Pre-LL), postoperative lumbar lordosis angle (Post-LL), Pre-LL minus Post-LL (Loss of LL), postoperative pelvic tilt (Post-PT), postoperative sacral slope, Pre-PI-LL mismatch (Pre-PI minus Pre-LL), and Post-PI-LL mismatch (Post-PI minus Post-LL). A 1-way analysis of variance (ANOVA) was performed with the aforementioned parameters, and binary logistic regression analysis was used to determine the relative risk factors. RESULTS: The 669 patients were followed-up for a mean of 2.7 ± 1.1 years (range, 2-4 years). Twenty-seven patients demonstrated fractures in the adjacent vertebral body after surgery. Analysis by 1-way ANOVA demonstrated that age, PI, Pre-LL, Post-LL, Loss of LL, Post-PI-LL mismatch, Post-PT, and osteoporosis were potential risk factors (all parameters, P < 0.001). Furthermore, binary logistic regression analysis showed that a large Loss of LL, osteoporosis, and old age were also risk factors for adjacent vertebral compression fractures. CONCLUSIONS: A greater Loss of LL, osteoporosis, and advanced age may be risk factors for fractures in the adjacent vertebral body of the fixed segment after lumbar fusion fixation.

Novel Surgical Strategy for Treating Osteoporotic Vertebral Fractures with Cord Compression.

OBJECTIVES: Treatment for osteoporotic vertebral fracture (OVF) with cord compression is challenging and it usually requires surgical interventions to decompress nerves and restore spinal sequences. To describe a novel surgical strategy for treating OVFs with cord compression. METHODS: This is a single-center retrospective analysis. The inclusion criteria were Frankel grade C-E, single level T10 -L2 . Between January 2008 and December 2016, a total of 56 OVF patients (47 females and nine males, with an average age of 72 years (66-88 years), comprising of eight grade C, 23 grade D, and 25 grade E patients) were enrolled. The treatment algorithm included preoperative evaluation by MRI, extension CT, and radiography to classify the OVFs as type 1.1 (reducible, stable; n = 13), type1.2 (reducible, unstable; n = 16), type 2 (irreducible; n = 19) or type 2M (modifier; n = 8). Vertebroplasty (VP)/kyphoplasty (KP) was applied in type 1.1. VP/KP with posterior fixation and posterolateral fusion was applied in type 1.2. And additional laminectomy/osteotomy was used in type 2, except in a modifier group (2M) where same procedure as applied for type 1.2 was used. VAS, ODI, Cobb angle, Frankel functional grade, and complications were recorded. RESULTS: Thirteen cases were classified as type 1.1, 16 cases as type 1.2, 19 cases as type 2, and eight cases as type 2M. The follow-up period was 38.9 months (range, 24-108 months). All patients were followed-up in at least 24 months, in which time four patients died, two patients were lost at the last follow-up, and 50 patients completed the full study. The total VAS and ODI improved from 8 (7, 9) and 75.5% (67.2%, 80.0%) preoperatively to 2 (1, 3) and 31% (24.0%, 37.0%) on conclusion, respectively (P < 0.01). The local kyphotic angle was corrected from 22.3° (17.1°, 33. 8°) preoperatively to 10.4° (6.4°, 15.3°) on conclusion (P < 0.01). Twenty-three patients had achieved neurological recovery on conclusion (42E, 8D, P < 0.01). Asymptotic cement leakage was observed in 17/56 cases (30.4%), 6/56 in the affected vertebra (10.7%), and 24/330 in the screw trajectory (7.3%). At 2 years postoperatively, 11 new VFs had occurred in nine patients (16.1%), including VFs in nine adjacent segments that all occurred within 1 year after surgery. No cement migration or implant failure was noted. CONCLUSION: The novel surgical strategy for treating OVFs with cord compression consists of the most tailored and least invasive treatment for each patient. The positive mid- and long-term clinical and radiological outcomes observed could represent a step forward in devising the proposed algorithm.

The cement leakage in cement-augmented pedicle screw instrumentation in degenerative lumbosacral diseases: a retrospective analysis of 202 cases and 950 augmented pedicle screws.

PURPOSE: To evaluate the incidence, type and risk factors of cement leakage (CL) with cement-augmented pedicle screw instrumentation (CAPSI) in degenerative lumbosacral disease. METHODS: Two hundred and two patients using a total of 950 cement-augmented screws were enrolled. CL was classified into three types: type S: leakage via segmental veins; type B: leakage via basivertebral veins; and type I: leakage via pedicle screw instrumentation to paravertebral soft tissue. The age, gender, operation stage (primary or later stage), body mass index, bone mineral density, the number and type of augmented screw, the position of the tip of screw (lateral or internal part of vertebral body), the position of screw (left or right side), the volume of bone cement, location of the augmented vertebra (lumbar or sacrum), the type of CL and complications were recorded. Binary logistic regression correlation was used to analyze risk factors of veins leakage (type S and type B). RESULTS: The CL was observed in 165 patients (81.68%) and 335 screws (35.26%), leakage types of S, B and I were seen in 255 (76.12%), 77 (22.99%), and 30 (8.96%) of screws, respectively. Besides, double or multiple routes of leakage were seen in 27 screws. Number of augmented screw was a risk factor for vein leakage (OR 0.58; 95% CI 0.44-0.77; P = 0.000). Furthermore, the doses of cement (OR 0.79; 95% CI 0.61-0.99; P = 0.038) and the position of screw (OR 0.39; 95% CI 0.29-0.53; P = 0.000) were identified as risk factors for type S, and the doses of bone cement (OR 0.37; 95% CI 0.25-0.54; P = 0.000) and the position of the tip of screw (OR 0.07; 95% CI 0.04-0.13; P = 0.000) were risk factors for type B. CONCLUSIONS: CAPSI bears a high risk of asymptomatic CL, with a higher rate of leakage into segmental veins and basivertebral veins. As is known, more augmented screws and larger doses of cement are risk factors for veins leakage (type S and type B), while the tip of screw approaching to the midline of the vertebral body is another risk factor to type B. Thus, the CL could be reduced by the amelioration of operative techniques and procedures. These slides can be retrieved under Electronic Supplementary Material.

[Establish mouse osteoblast -osteoclast cell co-culture system in a Transwell chamber].

OBJECTIVE: To establish osteoblast-osteoclast cell co-culture system in a Transwell chamber, and detect cell viability of osteoblasts and osteoclasts in system. METHODS: Osteoblast MC3T3-E1 and mouse monocytes RAW264.7 were cultivated in vitro. RANKL-induced mouse RAW264.7 monocytes differentiated into mature osteoclasts, osteoblast-osteoclast cell co-culture system was established in Transwell chamber. Cell activity of osteoblasts and osteoclasts were detected by CCK-8 experimenting, Alizarin Red staining, TRAP staining. The expression of OPG, ALP, RANKL, TGF-b1 gene and RANKL protein in osteoblast MC3T3-E1 were detected by PCR, Western-Blot methods. Also, the expression of RANK, NF-κB in gene and protein level in osteoclast were measured through the same method respectively. RESULTS: The co-culture system of Mouse MC3T3-E1 cells and RAW264.7 cell were established in Transwell chamber. Co-culture system affected cell division activities of osteoblasts and osteoclasts. Differentiation of osteoblasts were increased, while differentiation of osteoclast division were slight decreased under microscope observation. OPG (0.65±0.08) and ALP (0.16±0.01) gene expression of co-culture system were less than single culture OPG(1.00±0.08) and ALP (1.01±0.16); TGF-b1(4.42±0.21) and RANKL(4.12±1.04) of osteoblasts in co-culture system were higher than TGF-b1(1.00±0.10) and RANKL(1.00±0.09) under single culture. However, gene expression of RANK(0.63±0.06) and NF-κB(0.64±0.08) in co-culture system were decreased than RANK(1.00±0.08) and NF-κB(1.00±0.09), in single culture, and had significant differences. Similarly, protein expression of OPG(0.43±0.05) and NF-κB(0.59±0.05) of co-culture system were less than OPG(0.84±0.06) and NF-κB(1.13±0.03) of single culture. While RANKL protein expression (0.54±0.03)of co-culture system was more than single culture RANKL(0.31±0.03), and had statistically differences, which was in agreement of the trend of gene expression change. CONCLUSIONS: Co-culture system of mouse MC3T3-E1 cells and RAW264.7 cell was viable in Transwell chamber, and the activity of osteoblasts is higher than osteoclasts in co-culture system.