Learning curve of junior surgeons in robot-assisted pedicle screw placement: a comparative cohort study.

OBJECTIVE: Robot-assisted technology has been gradually applied to pedicle screw placement in spinal surgery. This study was designed to detailedly evaluate the learning curve of junior surgeons in robot-assisted spine surgery. METHODS: From December 2020 to February 2022, 199 patients requiring surgical treatment with posterior pedicle screw fixation were prospectively recruited into the study. The patients were randomized to the robot-assisted group (the RA group) or the conventional freehand group (the CF group). Under the senior specialist's supervision, pedicle screws were placed by two junior fellows without prior experience. Cumulative summation (CUSUM) analysis was performed on the learning curve of pedicle screw placement for performing quantitative assessment based on the time of screw insertion. RESULTS: In total, 769 and 788 pedicle screws were placed in the RA and CF groups. Compared with the CF group, the learning duration in the RA group was shorter in the upper thoracic region (57 vs. 70 screws), but longer in the lower thoracic (62 vs. 58 screws) and the lumbosacral region (56 vs. 48 screws). The slope of learning curve was lower in the RA group than in the CF group. The screw accuracy in the RA group was superior to that in the CF group, especially in upper thoracic region (89.4% vs. 76.7%, P < 0.001). This disparity of accuracy became wider in deformity cases. In the upper thoracic region, the mean placement time was 5.34 ± 1.96 min in the RA group and 5.52 ± 2.43 min in the CF groups, while in the lower thoracic and lumbosacral regions, the CF group's mean placement times were statistically shorter. Three screw-related neural complications occurred in the CF group. CONCLUSION: Robot-assisted technique has its advantages in the upper thoracic region and deformity cases, which is easier and safer to insert pedicle screws. The robot-assisted technique allowed a short learning curve for junior surgeons and exhibited consistently excellent results even in the early application period.

Is Complete Correction of Cervical Sagittal Malalignment Necessary During 4-Level Anterior Cervical Discectomy and Fusion Surgery in Patients With Kyphosis?

STUDY DESIGN: Retrospective analysis. OBJECTIVE: We investigated whether complete correction of cervical sagittal malalignment is necessary during 4-level anterior cervical discectomy and fusion (ACDF) in patients with kyphosis. METHODS: This retrospective study included 84 patients who underwent 4-level ACDF surgery at a university hospital between January 2010 and December 2015. Based on the degree of cervical lordosis correction, patients were categorized into the following groups: mild (0-10°), moderate (10-20°), and complete correction (>20°). The clinical outcomes, radiological parameters, and functional outcomes were analyzed. RESULTS: We observed no significant intergroup differences in the baseline characteristics. The cervical sagittal vertical axis (CSVA) correction loss at the final follow-up was lesser in the mild- and moderate- than in the complete-correction group. The spinocranial angle (SCA) and T1 slope (T1 S) were significantly higher in the moderate- and complete-correction groups than in the mild-correction group, 3 days postoperatively. The cervical proximal junctional kyphosis (CPJK), adjacent segment degeneration (ASD), and ASD following CPJK rates were higher in the complete-correction group. We observed no significant intergroup differences in postoperative complications; however, 5 patients showed internal fixation failure in the complete-correction group; 4 of these patients required reoperation. No significant intergroup difference was observed in the Japanese Orthopedic Association and neck disability index scores at any time point. CONCLUSIONS: A mild-to-moderate correction of cervical lordosis is superior to complete correction in patients with kyphosis who undergo 4-level ACDF because this approach is associated with lesser axial stress and CSVA correction loss.

Leptin accelerates BMSC transformation into vertebral epiphyseal plate chondrocytes by activating SENP1-mediated deSUMOylation of SIRT3.

Bone marrow mesenchymal stem cells (BMSCs) are capable of multidirectional differentiation, and engrafted BMSCs can be used to replace damaged chondrocytes for treatment of intervertebral disc disease. However, chondroblast differentiation of implanted BMSCs is inhibited by the anoxic environment of the articular cavity. Here, we found that leptin enhanced the transformation of BMSCs into chondrocytes under hypoxic conditions. BMSCs isolated from mice were cultured in medium supplemented with leptin under hypoxia. The expression of MFN1/2 and OPA1 were increased only in BMSCs cultured in an anoxic environment. In addition, in hypoxic environments cell energy metabolism relies on glycolysis regulated by leptin, rather than by mitochondrial oxidation. The expression of the de-SUMOylation protease SENP1 was elevated, leading to SIRT3-mediated activation of PGC-1α; these processes were regulated by CREB phosphorylation, and promoted mitochondrial fusion and cell differentiation. The chondrogenic activity of BMSCs isolated from SIRT3-knockout mice was lower than that of BMSCs isolated from wildtype mice. Implantation of SIRT3-knockout murine-derived BMSCs did not significantly improve the articular cartilage layer of the disc. In conclusion, the hypoxic microenvironment promoted BMSC differentiation into chondrocytes, whereas osteoblast differentiation was inhibited. SENP1 activated SIRT3 through the deSUMOylation of mitochondria and eliminated the antagonistic effect of SIRT3 acetylation on phosphorylation. When phosphorylation activity of CREB was increased, phosphorylated CREB is then transferred to the nucleus, affecting PGC-1α. This promotes mitochondrial fusion and differentiation of BMSCs. Leptin not only maintains chondrogenic differentiation homeostasis of BMSCs, but also provides energy for differentiation of BMSCs under hypoxic conditions through glycolysis.

C-type lectin domain-containing protein CLEC3A regulates proliferation, regeneration and maintenance of nucleus pulposus cells.

It is widely assumed that as connective tissue, the intervertebral disc (IVD) plays a crucial role in providing flexibility for the spinal column. The disc is comprised of three distinct tissues: the nucleus pulposus (NP), ligamentous annulus fibrous (AF) that surrounds the NP, and the hyaline cartilaginous endplates (CEP). Nucleus pulposus, composed of chondrocyte-like NP cells and its secreted gelatinous matrix, is critical for disc health and function. The NP matrix underwent dehydration accompanied by increasing fibrosis with age. The degeneration of matrix is almost impossible to repair, with the consequence of matrix stiffness and senescence of NP cells and intervertebral disc, suggesting the value of glycoproteins in extracellular matrix (ECM). Here, via database excavation and biological function screening, we investigated a C-type lectin protein, CLEC3A, which could support differentiation of chondrocytes as well as maintenance of NP cells and was essential to intervertebral disc homeostasis. Furthermore, mechanistic analysis revealed that CLEC3A could stimulate PI3K-AKT pathway to accelerate cell proliferation to further play part in NP cell regeneration.

Inclination of the small laminar slope angle leads to lumbar spinal stenosis due to hypertrophy of the ligamentum flavum.

OBJECTIVE: This study was designed to investigate the relationship between the laminar slope angle (LSA) and the lumbar disc degenerative grade, the cross-section area (CSA) of multifidus muscle, the muscle-fat index, and the thickness of the ligamentum flavum. METHODS: Retrospective analysis of 122 patients who were scheduled to undergo a lumbar operation for diagnoses associated with degenerative lumbar disease between January and December 2017. The L4-L5 disc grade was evaluated from preoperative sagittal T2-weighed magnetic resonance imaging of the lumber region; the CSA of the multifidus and muscle-fat index were measured at the L4 level, while the thickness of the ligamentum flavum was measured at the L4-L5 facet level from axis T2-weighed magnetic resonance imaging. The slope of the laminar was evaluated from preoperative three-dimensional computer tomography at the tip level of the facet joints and selected by the axis plane. Independent-sample T-tests were used to assess the association between age and measurement indices. RESULTS: Our results showed that age was positively connected with the LSA of L4 and L5 in different patients, although there was no significant difference between age and the difference of the two segment LSA. Partial correlation analysis, excluding the interference of age, revealed a strong negative relationship between the LSA of L4 and the thickness of the ligamentum flavum, irrespective of whether we considered the left or right. However, there was no correlation with lumbar disc degenerative grade, the CSA of the multifidus, and the muscle-fat index. CONCLUSION: The thickness of the ligamentum flavum showed changes with anatomical differences in the LSA, but not the lumbar disc degenerative grade, the CSA of the multifidus, and the muscle-fat index. A small change in LSA may cause large mechanical stress; this may be one of the causative factors responsible for lumbar spinal stenosis.

Circular RNA Expression Profile in Patients with Lumbar Spinal Stenosis Associated with Hypertrophied Ligamentum Flavum.

STUDY DESIGN: Sequencing and experimental analysis of the expression profile of circular RNAs (circRNAs) in hypertrophic ligamentum flavum (LFH). OBJECTIVES: The aim of this study was to identify differentially expressed circRNAs between LFH and nonhypertrophic ligamentum flavum tissues from lumbar spinal stenosis (LSS) patients. SUMMARY OF BACKGROUND DATA: Hypertrophy of the ligamentum flavum (LF) can cause LSS. circRNAs are important in various diseases. However, no circRNA expression patterns related to LF hypertrophy have been reported. METHODS: A total of 33 patients with LSS participated in this study. LF tissue samples were obtained when patients underwent decompressive laminectomy during surgery. The expression profile of circRNAs was analyzed by transcriptome high-throughput sequencing and validated with quantitative real-time polymerase chain reaction (PCR). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed for the differentially expressed circRNA-associated genes and related pathways. The connections between circRNAs and microRNAs were explored using Cytoscape. The role of hsa_circ_0052318 on LF cell fibrosis was assessed by analyzing the expression of collagen I and collagen III. RESULTS: The results showed that 2439 circRNAs of 4025 were differentially expressed between the LFH and nonhypertrophic ligamentum flavum tissues, including 1276 upregulated and 1163 downregulated circRNAs. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that these differentially expressed circRNAs functioned in biological processes, cellular components, and molecular functions. Autophagy and mammalian target of rapamycin were the top two signaling pathways affected by these circRNAs. Five circRNAs (hsa_circ_0021604, hsa_circ_0025489, hsa_circ_0002599, hsa_circ_0052318, and hsa_circ_0003609) were confirmed by quantitative real-time PCR. The network indicated a strong relationship between circRNAs and miRNAs. Furthermore, hsa_circ_0052318 overexpression decreased mRNA and protein expression of collagen I and III in LF cells from LFH tissues. CONCLUSION: This study identified circRNA expression profiles characteristic of hypertrophied LF in LSS patients, and demonstrated that hsa_circ_0052318 may play an important role in the pathogenesis of LF hypertrophy.Level of Evidence: N/A.

SIRT6 enhances telomerase activity to protect against DNA damage and senescence in hypertrophic ligamentum flavum cells from lumbar spinal stenosis patients.

Lumbar spinal stenosis (LSS) is a condition wherein patients exhibit age-related fibrosis, elastin-to-collagen ratio reductions, and ligamentum flavum hypertrophy. This study was designed to assess the relationship between SIRT6 and telomerase activity in hypertrophic ligamentum flavum (LFH) cells from LSS patients. We observed significant reductions in SIRT6, TPP1, and POT1 protein levels as well as increases in telomerase reverse transcriptase (TERT) levels and telomerase activity in LFH tissues relative to non- hypertrophic ligamentum flavum (LFN) tissues. When SIRT6 was overexpressed in these LFH cells, this was associated with significant increases in telomerase activity and a significant reduction in fibrosis-related protein expression. These effects were reversed, however, when telomerase activity was inactivated by hTERT knockdown in these same cells. SIRT6 overexpression was further found to reduce the frequency of senescence-associated ß-galactosidase (SA-ß-Gal)-positive LFH cells and to decrease p16, MMP3, and L1 mRNA levels and telomere dysfunction-induced foci (TIFs) in LFH cells. In contrast, hTERT knockdown-induced telomerase inactivation eliminated these SIRT6-dependent effects. Overall, our results indicate that SIRT6 functions as a key protective factor that prevents cellular senescence and telomere dysfunction in ligamentum flavum cells, with this effect being at least partially attributable to SIRT6-dependent telomerase activation.

MiR-874-3p plays a protective role in intervertebral disc degeneration by suppressing MMP2 and MMP3.

Intervertebral disc degeneration (IDD) is a spinal degenerative disease and one of the most important causes of musculoskeletal disability. Matrix metalloproteinase (MMP)-mediated extracellular matrix degradation is the core process of IDD. The regulators of MMPs in the intervertebral disc are still not fully known. In this study, using quantitative reverse transcription PCR, luciferase reporter assay, Western blotting, immunofluorescence, flow cytometry, and Cell Counting Kit-8 assay, we found that the miR-874-3p expression level was significantly decreased in IDD patients. MiR-874-3p could target and repress MMP2 and MMP3 expression in nucleus pulposus cells. These results could improve the understanding of IDD and provide a possible diagnostic marker and treatment candidate for IDD. The miR-874-3p/MMP2/MMP3 axis might also provide direction for future cancer and inflammation investigations.

Selective thoracolumbar/lumbar fusion for Syringomyelia-associated scoliosis: a case-control study with Lenke 5C adolescent idiopathic scoliosis.

BACKGROUND: Selective thoracolumbar/lumbar fusion technique was introduced to treat adolescent idiopathic scoliosis (AIS) patients with major thoracolumbar/lumbar curves. Theoretically, this surgical strategy could also be applied to syringomyelia patients. No previous study has specifically addressed the effectiveness of selective thoracolumbar/lumbar fusion for patients with syringomyelia-associated scoliosis. The aim of the study was to investigate the effectiveness of selective thoracolumbar/lumbar fusion for the surgical treatment of patients with syringomyelia-associated scoliosis. METHODS: From February 2010 to September 2016, 14 syringomyelia-associated patients with major thoracolumbar/lumbar curves were retrospectively reviewed. Besides, 30 Lenke 5C AIS patients were enrolled as a control group. Posterior selective thoracolumbar/lumbar fusion was performed for both groups. Patients' demographic, operative, radiological, and quality of life data were reviewed with follow-up. Intragroup comparisons were performed for each parameter. RESULTS: The two groups were matched by age, gender, curve characteristics, duration of follow-up, and all preoperative radiographic parameters except for thoracic kyphosis. After surgery, the average correction rate of the major thoracolumbar/lumbar curve was 82.2 ± 7.8% in the syringomyelia group, which was not significantly different from that of AIS group (82.5 ± 10.6%, P = 0.47). A similar improvement of unfused thoracic curve was observed between the two groups (50.1 ± 16.5% vs. 48.5 ± 26.9%, P = 0.29). During the follow-up, the correction effect of scoliosis was well maintained, without aggravation of the original neural symptoms or fresh permanent neurological deficits. Of note, the number of fusion levels was significantly larger in syringomyelia group than that in AIS group (7.6 ± 1.4 vs. 6.5 ± 1.2, P < 0.01). The average follow up was 47.6 months (36-81 months). CONCLUSION: Similar to AIS cases, syringomyelia-associated scoliosis can be effectively and safely corrected by selective thoracolumbar/lumbar fusion with satisfactory surgical outcomes. However, the syringomyelia group, on average, required an additional fused segment for treatment as compared to the AIS group (7.6 versus 6.5 in the AIS group).

Antinociceptive effect of intrathecal injection of miR-9-5p modified mouse bone marrow mesenchymal stem cells on a mouse model of bone cancer pain.

BACKGROUND: A growing body of studies have indicated that bone marrow mesenchymal stem cells (BMSCs) have powerful analgesic effects in animal models of bone cancer pain. Here, we explored the molecular mechanisms underlying how BMSCs alleviate pain sensation in a mouse model of bone cancer pain. METHODS: C3H/HeN adult male mice were used to generate a bone cancer pain model. BMSCs were isolated from mouse bone marrow, modified by transfection with microRNA-9-5p (miR-9-5p), and infused into the spinal cord. Spontaneous flinches, paw withdrawal latency, limb-use score, and weight-bearing score were used to assess pain-related behaviors. ELISA, RT-PCR, western blot, and luciferase assay were used to assess gene expressions. RESULTS: Our results show that miR-9-5p regulated the expression of both repressor element silencing transcription factor (REST) and µ-opioid receptors (MOR) by targeting REST in primary mouse BMSCs. Overexpression of miR-9-5p reversed the activation of inflammatory pathway in TNF-α- and IL-6-treated BMSCs. In addition, miR-9-5p modified BMSCs alleviated cancer pain in the sarcoma-inoculated mouse model. MiR-9-5p modified BMSCs suppressed cytokine expression in the spinal cord of sarcoma-inoculated mice by suppressing REST gene expression. CONCLUSIONS: Our results indicate that miR-9-5p modified BMSCs can relieve bone cancer pain via modulating neuroinflammation in the central nervous system, suggesting genetically modified BMSCs could be a promising cell therapy in pain management.

Polydatin induces apoptosis and autophagy via STAT3 signaling in human osteosarcoma MG-63 cells.

Polydatin, a natural product, is detected in many daily diets, such as grape juices and peanut. Autophagy regulation is recognized as a new potential strategy for cancer therapy, and previous studies demonstrated that polydatin showed remarkable anti-cancer ability. Nevertheless, the capability of polydatin to induce autophagy and its role in anti-osteosarcoma remains obscure. In this study, we investigated the anticancer effect of polydatin on human osteosarcoma cell line MG-63 and its underlying mechanism. Our results indicated that polydatin significantly inhibited proliferation of MG-63 cells in a dose- and time-dependent manner, and increased their apoptosis and autophagic flux. Further experiments showed that polydatin reduced the expression and phosphorylation (Y705) level of STAT3 (Signal transducer and activator of transcription 3), increased the expression of autophagy-related genes (Atg12, Atg14, BECN1, PIC3K3), and therewith triggered autophagic cell death in MG-63 cells. Of note, the cytotoxicity effect of polydatin was rescued by co-treatment with Colivelin (STAT3 activator), suggesting the dependency of MG-63 cells on STAT3 for survival in this process. Moreover, polydatin-triggered autophagy and apoptosis were remarkably reduced following exposure to autophagy inhibitor 3-methyladenine, while cell viability was increased. In conclusion, these data demonstrated that polydatin induced MG-63 cell death through inducing apoptosis, and autophagy which was mediated via the STAT3 signaling. Therefore, polydatin might be a potential clinical drug in the remedy of osteosarcoma.

LncRNA SNHG4 promotes tumour growth by sponging miR-224-3p and predicts poor survival and recurrence in human osteosarcoma.

OBJECTIVE: Accumulating data show that dysregulation of long noncoding RNAs (lncRNAs) acts a critical role in a variety of malignancies. Among these lncRNAs, small nucleolar RNA host genes (SNHGs) are associated with tumour growth and progression. But, the molecular mechanisms by which SNHG4 contributes to osteosarcoma remain undocumented. METHODS: The association between lncRNA SNHG4 expression and clinicopathologic characteristics and prognosis in patients with osteosarcoma was analysed by TCGA RNA-sequencing data. Cell viability and colony formation abilities were respectively assessed by MTT and colony formation assays. LncRNA SNHG4-specific binding with miR-224-3p was verified by bioinformatic analysis, luciferase gene report, and RNA immunoprecipitation assays. Regulation relationship between SNHG4 and miR-224-3p expression was further evaluated by the rescue experiments. RESULTS: The expression level of lncRNA SNHG4 was significantly elevated in osteosarcoma samples and cell lines as compared with the adjacent normal tissues, and SNHG4 high expression was associated with tumour size (TS) and poor prognosis in patients with osteosarcoma. Knockdown of SNHG4 suppressed cell viability and invasive potential, whereas ectopic SNHG4 expression displayed the opposite effects. Moreover, we found that lncRNA SNHG4 acted as a sponge of miR-224-3p, and miR-224-3p mimic reversed SNHG4 induced tumour-promoting effects in osteosarcoma cells. The expression of miR-224-3p depicted a negative correlation with SNHG4 in osteosarcoma samples and miR-224-3p low expression was associated with TS and poor survival in patients with osteosarcoma. CONCLUSION: Our findings demonstrated that LncRNA SNHG4 promoted tumour growth by sponging miR-224-3p and represented a poor prognostic factor in patients with osteosarcoma.

miR-149-5p inhibits cell growth by regulating TWEAK/Fn14/PI3K/AKT pathway and predicts favorable survival in human osteosarcoma.

MicroRNAs (miRNAs) as small non-coding RNAs act as either tumor suppressors or oncogenes in human cancers, of which miR-149-5p (miR-149) is involved in tumor growth and metastasis, but its role and molecular mechanisms underlying osteosarcoma growth are poorly understood. The correlation of miR-149 expression with clinicopathological characteristics and prognosis in patients with sarcoma was analyzed by The Cancer Genome Atlas (TCGA) RNA-sequencing data. Osteosarcoma cell growth affected by miR-149 was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation assays. As a result, we found that the expression level of miR-149 was markedly downregulated in human sarcoma samples and were negatively associated with tumor size, acting as an independent prognostic factor for overall survival of the sarcoma patients. Restoration of miR-149 expression suppressed osteosarcoma cell growth, while its knockdown reversed these effects. Furthermore, we identified TNFRSF12A (TNF receptor superfamily member 12A), also called fibroblast growth factor-inducible 14 (Fn14) as a direct target of miR-149, and TNFRSF12A and its ligand TNFSF12 (TNF superfamily member 12), also called tumor necrosis factor-related weak inducer of apoptosis (TWEAK), were both negatively correlated with miR-149 expression in sarcoma samples. Knockdown of TNFRSF12A suppressed cell growth, but its overexpression weakened the antiproliferative effects of miR-149 via the PI3K/AKT (AKT serine/threonine kinase) signaling pathway. Altogether, our findings show that miR-149 functions as a tumor suppressor in osteosarcoma via inhibition of the TWEAK-Fn14 axis and represents a potential therapeutic target in patients with osteosarcoma.

Leptin differentially regulates endochondral ossification in tibial and vertebral epiphyseal plates.

Longitudinal bone growth is governed by a complex network of endocrine signals including leptin. In mouse, leptin deficiency leads to distinct phenotypes in bones of the limb and spine, suggesting the appendicular and axial skeletons are subject to differential regulation by leptin. We established primary cultures for the chondrocytes from tibial and vertebral epiphyseal plates. Cellular proliferation and apoptosis were analyzed for the chondrocytes that had been treated with various concentrations of leptin. Crucial factors for chondrocyte proliferation and differentiation, such as BMP7 and Wnt3, were measured in the cells treated with leptin alone or in combination with pharmacological inhibitors of STAT and ERK signaling pathways. Primary culture of tibial epiphyseal plate chondrocytes has greater proliferating capability compared with that of vertebral epiphyseal plate chondrocytes. Leptin could promote the proliferation of tibial epiphyseal plate chondrocytes, while its effect on vertebral epiphyseal plate chondrocytes was inhibitory. Consistently, apoptosis is inhibited in tibial but promoted in vertebral epiphyseal plate chondrocytes by leptin. Importantly, leptin differentially modulates chondrogenic signaling pathways in tibial and vertebral epiphyseal chondrocytes through STAT and ERK pathways. Leptin differentially regulates chondrogenic proliferation and differentiation in appendicular and axial regions of the skeletons. The signaling pathways in these two regions are also distinct and subject to differential regulation by leptin through the STAT pathway in tibial epiphyseal plate chondrocytes but through the ERK pathway in vertebral epiphyseal plate chondrocytes. Therefore, the regulation of leptin is multi-faceted in the distinct anatomical regions of the skeleton. Knowledge gained from this system will provide insights into the pathophysiological causes for the diseases related to bone development and metabolism.

Effects of Low Bone Mineral Status on Biomechanical Characteristics in Idiopathic Scoliotic Spinal Deformity.

BACKGROUND: Low bone mass in patients with adolescent idiopathic scoliosis has been well reported. Poor bone quality was regarded as a new and unique prognostic factor in aggravating curve progression. However, the potential biomechanical correlation between them remains unclear. METHODS: Three-dimensional finite element models of idiopathic scoliotic spine with different bone mineral status were created for axial loading simulation. An axial load of 3 different body weights was applied on different bone mineral mass models. The mechanical responses of the vertebral cortical and cancellous bone, facet joints, end plate, and intervertebral disc were analyzed. RESULTS: Accompanied with the low bone mineral status, thoracic scoliosis produced asymmetric and higher stress in the cortical bone, lumbar facet joints, and end plate at the concave side of the thoracic structure curve. Stress increased in the disc at the apex of the scoliosis, whereas it mildly decreased in the L4-5 and L5-S1 disc. Body weight gain increased the stress in scoliotic spine structures in all bone mineral statues. CONCLUSIONS: Biomechanical simulations indicated that low bone mineral mass might aggravate curve progression and induce more serious lumbar compensatory scoliosis in patients with adolescent idiopathic scoliosis. Weight gain was also a risk factor for curve progression.

Regulation of osteogenic differentiation by DNA methylation of the dishevelled gene in bone marrow mesenchymal stem cells.

Bone marrow mesenchymal stem cells (BMSCs) are stem cells with multidirectional differentiation potential, which can be used as seed cells to repair and reconstruct many types of tissues and organs following injury or disease. Osteogenic differentiation involves a variety of pathway and factors, including cytokines, growth factors, and hormones. In the present study, we investigated the potential role of Dishevelled in osteogenic differentiation of BMSCs in induction medium containing the methyltransferase inhibitor 5-aza-2'-deoxycytidine. The expression of Dishevelled was analyzed using the reverse transcriptase-polymerase chain reaction (RT-PCR) and a Western blot. The methylation degree of the CpG island in the promoter region of the Dishevelled gene was analyzed, and protein expression levels of Wnt, Glycogen synthase kinase-3 (GSK3), axin, Dishevelled, and ß-catenin were increased after the addition of the methyltransferase inhibitor. The expression of Dishevelled increased in accordance with the differentiation of osteoblasts, and the degree of methylation of the promoter affected its expression level. In conclusion, regulating the methylation degree of the Dishevelled gene promoter region appears to influence the expression of Dishevelled and therefore the osteogenic differentiation of BMSCs.

Effect of Asymmetric Tension on Biomechanics and Metabolism of Vertebral Epiphyseal Plate in a Rodent Model of Scoliosis.

OBJECTIVE: To investigate the effect of asymmetric tension on idiopathic scoliosis (IS) and to understand its pathogenic mechanism. METHODS: The rodent model of scoliosis was established using Sprague-Dawley rats with left rib-tethering from T6 to T12 , tail and shoulder amputation, and high-cage feeding. Vertebrae epiphyseal cartilage plates were harvested from the convex and concave sides. To analyze differences on the convex and concave sides, finite element analysis was carried out to determine the mechanical stress. Protein expression on epiphyseal cartilage was evaluated by western blot. Micro-CT was taken to evaluate the bone quality of vertebral on both sides. RESULTS: Scoliosis curves presented in X-ray radiographs of the rats. Finite element analysis was carried out on the axial and transverse tension of the spine. Stresses of the convex side were -170.14, -373.18, and -3832.32 MPa (X, Y, and Z axis, respectively), while the concave side showed stresses of 361.99, 605.55, and 3661.95 MPa. Collagen type II, collagen type X, Sox 9, RunX2, VEGF, and aggrecan were expressed significantly more on the convex side (P < 0.05). There was asymmetric expression of protein on the epiphyseal cartilage plate at molecular level. Compared with the convex side, the concave side had significantly lower value in the BV/TV and Tb.N, but higher value in the Tb.Sp (P < 0.05). There was asymmetry of bone quality in micro-architecture. CONCLUSIONS: In this study, asymmetric tension contributed to asymmetry in protein expression and bone quality on vertebral epiphyseal plates, ultimately resulting in asymmetry of anatomy. In addition, asymmetry of anatomy aggravated asymmetric tension. It is the first study to show that there is an asymmetrical vicious circle in IS.

RNA sequencing identifies gene expression profile changes associated with ß-estradiol treatment in U2OS osteosarcoma cells.

This study was conducted to identify gene expression profile changes associated with ß-estradiol (E2) treatment in U2OS osteosarcoma cells by high-throughput RNA sequencing (RNA-seq). Two U2OS cell samples treated with E2 (15 µmol/L) and two untreated control U2OS cell samples were subjected to RNA-seq. Differentially expressed genes (DEGs) between the groups were identified, and main biological process enrichment was performed using gene ontology (GO) analysis. A protein-protein interaction (PPI) network was constructed using Cytoscape based on the Human Protein Reference Database. Finally, NFKB1 expression was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). The map ratios of the four sequenced samples were >65%. In total, 128 upregulated and 92 downregulated DEGs were identified in E2 samples. After GO enrichment, the downregulated DEGs, such as AKT1, were found to be mainly enriched in cell cycle processes, whereas the upregulated DEGs, such as NFKB1, were involved in the regulation of gene expression. Moreover, AKT1 (degree =117) and NFKB1 (degree =72) were key nodes with the highest degrees in the PPI network. Similarly, the results of qRT-PCR confirmed that E2 upregulated NFKB1 expression. The results suggest that E2 upregulates the expression of NFKB1, ATF7IP, and HDAC5, all of which are involved in the regulation of gene expression and transcription, but downregulates that of TCF7L2, ALCAM, and AKT, which are involved in Wnt receptor signaling through ß-catenin and morphogenesis in U2OS osteosarcoma cells.

Autophagic degradation of FOXO3a represses the expression of PUMA to block cell apoptosis in cisplatin-resistant osteosarcoma cells.

Autophagy and apoptosis are the two major modes of cell death, and autophagy usually inhibits apoptosis. The current understanding has shown that there is a complex crosstalk between the components of these two pathways. Here, we describe a transcriptional mechanism that links autophagy to apoptosis. We show that the cisplatin-resistant MG63-R12 and U2OS-R5 osteosarcoma sublines, in comparison to their parental MG63 and U2OS cells, respectively, exhibit increased autophagy but decreased apoptosis levels after treatment with cisplatin. We then used a microarray assay to examine the gene expression changes in these two cisplatin-resistant sublines and found that the expression of the transcription factor FOXO3a was dramatically decreased. Pharmacological treatment with either 3-methyladenine to inhibit autophagy or with rapamycin to activate autophagy in these two cisplatin-resistant sublines resulted in the accumulation or degradation of FOXO3a, respectively. Ectopic expression of FOXO3a in MG63-R12 and U2OS-R5 cells significantly enhanced cell sensitivity to cisplatin through a mechanism in which FOXO3a directly binds to the PUMA promoter and activates its expression, as well as its downstream event, the intrinsic apoptosis pathway. Importantly, this overexpression resulted in tumor growth inhibition in vivo. In conclusion, our results provide new insights into the molecular link between autophagy and apoptosis that involves a FOXO3a-mediated transcriptional mechanism. Importantly, our results may facilitate the development of therapeutic strategies for osteosarcoma patients who have become resistant to cisplatin therapy.

Leptin differentially regulates chondrogenesis in mouse vertebral and tibial growth plates.

BACKGROUND: Leptin plays an important role in mediating chondrogenesis of limb growth plate. Previous studies suggest that bone structures and development of spine and limb are different. The expression of Ob-Rb, the gene that encodes leptin receptors, is vertebral and appendicular region-specific, suggesting the regulation of leptin on VGP and TGP chondrogenesis may be very different. The aim of the present study was to investigate the differential regulation of leptin on the chondrogenesis of vertebral growth plate (VGP) and tibial growth plate (TGP). METHODS: We compared the VGP and TGP from wild type (C57BL/6) and leptin-deficient (ob/ob) mice. We then generated primary cultures of TGP and VGP chondrocytes. By treating the primary cells with different concentrations of leptin in vitro, we analyzed proliferation and apoptosis of the primary chondrocytes from TGP and VGP. We further measured expression of chondrogenic-related genes in these cells that had been incubated with different doses of leptin. RESULTS: Leptin-deficient mice of 8-week-old had shorter tibial and longer vertebral lengths than the wide type mice. Disturbed columnar structure was observed for TGP but not for VGP. In primary chondrocyte cultures, leptin inhibited VGP chondrocyte proliferation but promoted their apoptosis. Collagen IIA and aggrecan mRNA, and the protein levels of proliferation- and chondrogenesis-related markers, including PCNA, Sox9, and Smad4, were downregulated by leptin in a dose-dependent manner. In contrast, leptin stimulated the proliferation and chondrogenic differentiation of TGP chondrocytes at physiological levels (i.e., 10 and 50 ng/mL) but not at high levels (i.e., 100 and 1000 ng/mL). CONCLUSION: Leptin exerts a stimulatory effect on the proliferation and chondrogenic differentiation of the long bone growth plate but an inhibitory effect on the spine growth plate. The ongoing study will shed light on the regulatory mechanisms of leptin in bone development and metabolism.