Phosphorylated heat shock protein 27 improves the bone formation ability of osteoblasts and bone marrow stem cells from patients with adolescent idiopathic scoliosis.

Background: Adolescent idiopathic scoliosis (AIS) is a scoliotic deformity of unknown etiology that occurs during adolescent development. Abnormal bone metabolism is closely related to AIS, but the cause is uncertain. Recent studies have shown that heat shock protein 27 (HSP27) and its phosphorylation (pHSP27) play important roles in bone metabolism. However, whether HSP27 and pHSP27 are involved in abnormal bone metabolism in AIS is unclear. Methods: Osteoblasts (OBs) and bone marrow stem cells (BMSCs) were extracted from the facet joints and bone marrow of AIS patients and controls who underwent posterior spinal fusion surgery. The expression levels of HSP27 and pHSP27, as well as the expression levels of bone formation markers in OBs from AIS patients and controls, were examined by quantitative real-time PCR (qRT-PCR) and Western blotting. The mineralization ability of OBs from AIS patients and controls was analyzed by alizarin red staining after osteogenic differentiation. Heat shock and thiolutin were used to increase the levels of pHSP27 in OBs, and the levels of bone formation markers were also investigated. In addition, the levels of pHSP27 and the bone formation ability of BMSCs from AIS patients and controls were investigated after heat shock treatment. Results: Lower pHSP27 levels and impaired osteogenic differentiation abilities were observed in the OBs of AIS patients than in those of controls. Thiolutin increased HSP27 phosphorylation and increased the mRNA levels of SPP1 and ALPL in OBs from AIS patients. Heat shock treatment increased SPP1 and HSP27 mRNA expression, pHSP27 levels, OCN expression, and mineralization ability of both OBs and BMSCs from AIS patients. Conclusion: Heat shock treatment and thiolutin can increase the levels of pHSP27 and further promote the bone formation of OBs and BMSCs from AIS patients. Therefore, decreased pHSP27 levels may be associated with abnormal bone metabolism in AIS patients.

Upregulated estrogen receptors impairs myogenesis and elevates adipogenesis related factor levels in the paravertebral muscles of patients with idiopathic scoliosis.

The prevalence of idiopathic scoliosis (IS) is 2-3% worldwide and is more common in girls. Estrogen receptors (ERs) is supposed to be related to sex differences and development of IS. Meanwhile, paravertebral muscle (PVM) abnormalities play important roles in the pathogenesis of IS. But the changes of ERs between the PVMs from IS patients and controls, and the mechanism by which ERs may affect IS patients remain unclear. Thus, the expression levels of ERs, myogenesis regulator (MYOG) and adipogenesis related factors (CEBPA, PPARγ, FABP4), as well as morphological changes in the PVMs and primary skeletal muscle mesenchymal progenitor cells (hSM-MPCs) of IS patients and controls were investigated. Increased expression levels of ERs and CEBPA, PPARγ, FABP4, together with severe myofiber necrosis and fat infiltration, were found in the PVMs of IS patients. Meanwhile, upregulated ERs, FABP4 and CEBPA, downregulated MYOG and impaired myogenesis were also revealed in the hSM-MPCs of IS patients compared with those of controls. Upregulation of ERs inhibited myogenesis but increased expression of CEBPA and FABP4 in C2C12 myoblasts. Nevertheless, treatment of ER antagonist increased expression of MYOG, enhanced myogenesis and decreased expression of CEBPA and FABP4 in skeletal muscle cells of IS patients. Therefore, our study suggested that PVMs specific upregulation of ERs could impair myogenesis and increase the expression of adipogenesis related factors, further leading to PVMs abnormalities in IS patients.

Impaired autophagy activity-induced abnormal differentiation of bone marrow stem cells is related to adolescent idiopathic scoliosis osteopenia.

BACKGROUND: Osteopenia has been well documented in adolescent idiopathic scoliosis (AIS). Bone marrow stem cells (BMSCs) are a crucial regulator of bone homeostasis. Our previous study revealed a decreased osteogenic ability of BMSCs in AIS-related osteopenia, but the underlying mechanism of this phenomenon remains unclear. METHODS: A total of 22 AIS patients and 18 age-matched controls were recruited for this study. Anthropometry and bone mass were measured in all participants. Bone marrow blood was collected for BMSC isolation and culture. Osteogenic and adipogenic induction were performed to observe the differences in the differentiation of BMSCs between the AIS-related osteopenia group and the control group. Furthermore, a total RNA was extracted from isolated BMSCs to perform RNA sequencing and subsequent analysis. RESULTS: A lower osteogenic capacity and increased adipogenic capacity of BMSCs in AIS-related osteopenia were revealed. Differences in mRNA expression levels between the AIS-related osteopenia group and the control group were identified, including differences in the expression of LRRC17 , DCLK1 , PCDH7 , TSPAN5 , NHSL2 , and CPT1B . Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed several biological processes involved in the regulation of autophagy and mitophagy. The Western blotting results of autophagy markers in BMSCs suggested impaired autophagic activity in BMSCs in the AIS-related osteopenia group. CONCLUSION: Our study revealed that BMSCs from AIS-related osteopenia patients have lower autophagic activity, which may be related to the lower osteogenic capacity and higher adipogenic capacity of BMSCs and consequently lead to the lower bone mass in AIS patients.

Abnormal TNS3 gene methylation in patients with congenital scoliosis.

BACKGROUND: Congenital scoliosis (CS) is a congenital deformity of the spine resulting from abnormal and asymmetrical development of vertebral bodies during pregnancy. However, the etiology and mechanism of CS remain unclear. Epigenetics is the study of heritable variations in gene expression outside of changes in nucleotide sequence. Among these, DNA methylation was described first and is the most characteristic and most stable epigenetic mechanism. Therefore, in this study, we aim to explore the association between genome methylation and CS which are not been studied before. METHODS: Two pairs of monozygotic twins were included, with each pair involving one individual with and one without CS. Agilent SureSelect XT Human Methyl-Sequencing was used for genome methylation sequencing. MethylTarget was used to detect methylation levels in target regions. Immunohistochemistry was performed to visualize expression of associated genes in candidate regions. RESULTS: A total of 75 differentially methylated regions were identified, including 24 with an increased methylation level and 51 with a decreased methylation level in the CS group. Nine of the differentially methylated regions were selected (TNS3, SEMAC3, GPR124, MEST, DLK1, SNTG1, PPIB, DEF8, and GRHL2). The results showed that the methylation level of the promoter region of TNS3 was 0.72 ± 0.08 in the CS group and 0.43 ± 0.06 in the control group (p = 0.00070 < 0.01). There was no significant difference in the degree of methylation of SEMAC3, GPR124, MEST, DLK1, SNTG1, PPIB, DEF8, or GRHL2 between the two groups. Immunohistochemistry showed significantly decreased TNS3 expression in the cartilage of the articular process in CS (CS: 0.011 ± 0.002; control: 0.018 ± 0.006, P = 0.003 < 0.01). CONCLUSION: Compared with the control group, high-level methylation of the TNS3 promoter region and low TNS3 expression in the cartilage layer of the articular process characterize CS. Thus, DNA methylation and TNS3 may play important roles in the pathogenesis of CS.

Spinous Process Combined With a Titanium Mesh Cage as a Bone Graft in the Stability Reconstruction of Lumbar or Lumbosacral Spinal Tuberculosis.

Background: Autogenous bone grafts, such as iliac bone or rib struts, have been used in the anterior reconstruction of spinal tuberculosis (STB) and have their own benefits and limitations. Here, we introduced a new method, the spinous process (SP), combined with a titanium mesh cage (TMC) as a bone graft in the stability reconstruction of lumbar or lumbosacral STBs. By retrospectively comparing patients who received SP+TMC to traditional TMC bone grafts or allogeneic bone grafts in terms of safety, efficacy and cost-effectiveness, we aimed to evaluate whether SP+TMC could be a possible alternative method. Methods: From 2010 to 2018, 69 patients who underwent one-stage posterior debridement with grafts and internal fixation within a single lumbar or lumbosacral segment were included in this study. Twelve patients who received SP combined with a TMC (SP+TMC, group A), 30 patients who received a TMC only (group B), and 27 patients who received allografts (group C) were included. Measurements including operative time, blood loss, length of hospital stay, visual analog scale (VAS) score, Oswestry Disability Index (ODI), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), American Spinal Injury Association Impairment (ASIA) grade, final follow-up (FFU) duration and postoperative complications were recorded. Radiological measurements, including the number of segments fixated, the number of pedicle screws used, the Cobb angle, pelvic parameters, and the bony fusion time, were reviewed. All outcomes were analyzed using SPSS 25. Results: We found that the SP+TMC group had fewer fixation segments, fewer pedicle screws implanted, a shorter operative time, reduced blood loss, and a considerably lower hospital cost than allografts. In addition, the TMC group had a comparable clinical outcome with the TMC group regarding lower economic cost. Conclusion: Our study demonstrates that compared to a TMC or allograft, the use of SP combined with a TMC as a bone graft is an effective and reliable approach for the surgical management of one-level lumbar or lumbosacral spinal tuberculosis, leading to effective restoration of spinal stability. Furthermore, this approach is a cost-effective structural bone grafting method, especially for patients in developing countries.

Preoperative Halo-Femoral Traction With Posterior Surgical Correction for the Treatment of Extremely Severe Rigid Congenital Scoliosis (Cobb Angle >120°).

INTRODUCTION: To evaluate the effectiveness and safety of preoperative halo-femoral traction (HFT) with posterior surgical correction for the treatment of extremely severe rigid congenital scoliosis (>120°). METHODS: We reviewed the records of all patients with extremely severe rigid congenital scoliosis (>120°) treated with preoperative HFT from 2010 through 2018. Radiographic measurements were performed. The period of traction, blood loss, operation time, complications, and pulmonary function test results were recorded. RESULTS: A total of 11 patients were included in the study. All patients underwent preoperative HFT with posterior surgical correction. The mean preoperative main curve Cobb angle was 127.9° ± 4.4°, and the average correction rate was 33.5% posttraction, 54.8% postoperation, and 55.3% at the latest follow-up. Pulmonary function improved significantly after traction, and forced vital capacity and forced expiratory volume in 1 second increased from 34.7% and 33.4% to 48.1% and 48.5%, respectively. Only one patient experienced halo pin infection during HFT. No patients experienced permanent neurologic deficits or death. CONCLUSION: For extremely severe rigid congenital scoliosis with a Cobb angle greater than 120°, preoperative HFT with posterior surgical correction offers an effective and safe corrective option. The perioperative complication rate can be partially reduced. STUDY DESIGN: Retrospective study.

Nontargeted Metabolomic Analysis of Plasma Metabolite Changes in Patients with Adolescent Idiopathic Scoliosis.

OBJECTIVE: Adolescent idiopathic scoliosis (AIS) is a relatively common spinal rotation deformity, and the pathogenesis of AIS is accompanied by metabolic dysfunction and changes in biochemical factors. In this study, plasma metabolite changes in AIS patients were analyzed based on nontargeted metabolomics to provide new insights for clarifying functional metabolic abnormalities in AIS patients. METHODS: Clinical indexes and blood samples were collected from 12 healthy subjects and 16 AIS patients. Metabolomics was used to analyze the changes in metabolites in plasma samples. The correlation between plasma metabolites and clinical indexes was analyzed by the Spearman rank correlation coefficient. RESULTS: Analysis of clinical data showed that the body weight, body mass index (BMI), and bone mineral density (BMD) index of the AIS group significantly decreased, while the blood phosphorus and Cobb angles increased significantly. Metabolomic analysis showed significant changes in 72 differential metabolites in the plasma of the AIS group, mainly including organooxygen compounds, carboxylic acids and derivatives, fatty acyls, steroids and steroid derivatives, and keto acids and derivatives. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway showed that arginine biosynthesis, D-glutamine and D-glutamate metabolism, alanine, aspartate and glutamate metabolism, and citrate cycle (TCA cycle) were significantly enriched in the AIS and healthy groups. Spearman rank correlation coefficient analysis showed that the plasma metabolites C00026 (oxoglutarate), C00062 (L-arginine, arginine), C01042 (N-acetylaspartate), and C00158 (citrate) were significantly correlated with clinical indexes in AIS patients. In the healthy group, the plasma metabolites C00122 (fumarate), C00025 (glutamate and L-glutamic acid) and C00149 (malate, L-malic acid) were significantly correlated with clinical indexes, while C00624 (N-acetylglutamate) was not significantly correlated with the clinical indexes. CONCLUSION: The occurrence of AIS led to changes in clinical indexes and plasma metabolites. Plasma biomarkers and functional metabolic pathways were correlated with clinical indexes, which might provide new insights for the diagnosis and treatment of AIS.

Altered Circulating Cell-free Mitochondrial DNA of Patients with Congenital Scoliosis.

STUDY DESIGN: Case-control study. OBJECTIVE: The aim of this study was to estimate the relationship between circulating cell-free DNA (ccf DNA) and clinical parameters of patients with congenital scoliosis (CS). SUMMARY OF BACKGROUND DATA: CS is a complex spinal deformity characteristic of congenital vertebral malformations. Although numerous studies have centered on the etiology of CS, the cause of CS remains unclear. Previously, we reported that circulating cell-free DNA (ccf DNA) is altered in adolescent idiopathic scoliosis (AIS). However, the relationship between ccf DNA and the clinical parameters of patients with CS remains unclear. METHODS: The plasma of peripheral blood from 35 patients with CS and 32 age-matched controls was collected for ccf DNA analysis. Quantitative PCR was used to detect ccf n-DNA and ccf mt-DNA levels, and correlation analyses between ccf n-DNA and ccf mt-DNA levels were conducted. Receiver-operating characteristic (ROC) curves were used to analyze the sensitivity and specificity of ccf n-DNA and ccf mt-DNA levels to different characteristics. RESULTS: The plasma ccf mt-DNA levels of both ND1 and CYTC were significantly decreased in patients with CS compared with levels in controls both in total and by sex, whereas the plasma ccf n-DNA levels showed no significant difference. There is no difference in both ccf mt-DNA and ccf n-DNA between S-SDV and M-SDV according to The International Consortium for Vertebral Anomalies and Scoliosis (ICVAS) classification. The ROC curve analyses showed a reliable sensitivity and specificity of CS predicted by ccf mt-DNA levels in total but failed to distinguish different ICVAS types. CONCLUSION: Significantly decreased plasma ccf mt-DNA levels were observed in patients with CS compared with those in controls. Although this finding has limited significance for clinical practice, it indicates that ccf mt-DNA may predict the onset or development of CS. Further studies should focus on the role of ccf mt-DNA in embryo development and whether ccf mt-DNAs could be considered as a marker for prenatal screening in development disorder like CS.Level of Evidence: 4.

Analysis of the corrective contribution of strong halo-femoral traction in the treatment of severe rigid nonidiopathic scoliosis.

INTRODUCTION: Strong halo-femoral traction has been widely used in the field of severe rigid scoliosis correction. The objective of this study was to analyze the corrective contribution of strong halo-femoral traction in the treatment of severe rigid nonidiopathic scoliosis and discuss its meaning. MATERIAL AND METHODS: A retrospective review was performed for patients with severe rigid nonidiopathic scoliosis who were treated with halo-femoral traction in our center from December 2008 to December 2015. All cases underwent halo-femoral traction for 2 to 4 weeks before a one-stage posterior operation, and the absolute and relative contribution rates of each orthopedic factor (bending, fulcrum, traction, surgery) were analyzed. RESULTS: A total of 38 patients were included (15 males and 23 females), with a mean age of 16.4 ± 3.73 years (10-22 years) and follow-up of 55.05 ± 6.63 months (range 40-68 months). The etiology was congenital in 17 patients, neuromuscular in 14 patients, neurofibromatosis-1 in 3 patients, and Marfan syndrome in 2 patients. Congenital high scapular disease with scoliosis was found in 2 patients. The mean coronal Cobb angle of the major curve was 97.99° ± 11.47° (range 78°-124°), with a mean flexibility of 15.68% ± 6.65%. The absolute contribution rate (ACR) of bending was 27.26% ± 10.16%, the ACR of the fulcrum was 10.91% ± 2.50%, the ACR of traction was 32.32% ± 11.41%, and the ACR of surgery was 29.50% ± 9.70%. A significant difference in correction was noted between the ACRs of traction and the fulcrum (P < 0.05). DISCUSSION: Strong halo-femoral traction plays a relatively significant role in the treatment of severe rigid nonidiopathic scoliosis while decreasing the risk of operation, and it is a safe and effective method for the treatment of severe rigid nonidiopathic scoliosis.

Coding Variants Coupled With Rapid Modeling in Zebrafish Implicate Dynein Genes, dnaaf1 and zmynd10, as Adolescent Idiopathic Scoliosis Candidate Genes.

Adolescent idiopathic scoliosis (AIS) is the most common pediatric spine disorder affecting ∼3% of children worldwide. Human genetic studies suggest a complex polygenic disease model for AIS with large genetic and phenotypic heterogeneity. However, the overall genetic etiology of AIS remains poorly understood. To identify additional AIS susceptibility loci, we performed whole-exome sequencing (WES) on a cohort of 195 Southern Chinese AIS patients. Bioinformatics analysis identified 237 novel rare variants associated with AIS, located in 232 new susceptibility loci. Enrichment analysis of these variants revealed 10 gene families associated with our AIS cohort. We screened these gene families by comparing our candidate gene list with IS candidate genes in the Human Phenotype Ontology (HPO) database and previous reported studies. Two candidate gene families, axonemal dynein and axonemal dynein assembly factors, were retained for their associations with ciliary architecture and function. The damaging effects of candidate variants in dynein genes dnali1, dnah1, dnaaf, and zmynd10, as well as in one fibrillin-related gene tns1, were functionally analyzed in zebrafish using targeted CRISPR/Cas9 screening. Knockout of two candidate genes, dnaaf1 or zmynd10, recapitulated scoliosis in viable adult zebrafish. Altogether, our results suggest that the disruption of one or more dynein-associated factors may correlate with AIS susceptibility in the Southern Chinese population.

Dysregulated Bone Metabolism Is Related to High Expression of miR-151a-3p in Severe Adolescent Idiopathic Scoliosis.

Adolescent idiopathic scoliosis (AIS) is a common complex disease, and bone homeostasis plays an important role in its pathogenesis. Recent advances in epigenetic research show that dysregulated miRNAs may participate in the development of orthopedic diseases and AIS. The aim of this study was to detect differentially expressed miRNAs in severe AIS and elucidate the mechanism of miRNA deregulation in the pathogenesis of AIS. In the present study, miRNA expression profiles were detected in severe and mild AIS patients as well as healthy controls by miRNA sequencing. Candidate miRNAs were validated in a larger cohort. Primary osteoblasts from severe AIS patients were extracted and isolated to determine the effect of the candidate miRNAs on bone metabolism. Finally, we determined the methylation level in primary osteoblasts from severe AIS patients. The result showed that miR-151a-3p was overexpressed in severe AIS patients. Reduced GREM1 expression was observed in primary osteoblasts from severe AIS patients. miR-151a-3p directly inhibited GREM1 in primary osteoblasts. Relatively lower methylation levels were detected in primary osteoblasts from severe AIS patients. In conclusion, our study revealed that plasma miR-151a-3p levels may serve as a biomarker for severe AIS. Overexpression of miR-151a-3p may interrupt bone homeostasis via inhibiting GREM1 expression. Our result may provide a new biomarker for the early detection of AIS and increase our understanding of the pathogenesis of AIS.

High methylation of lysine acetyltransferase 6B is associated with the Cobb angle in patients with congenital scoliosis.

BACKGROUND: The etiology of congenital scoliosis (CS) is complex and uncertain. Abnormal DNA methylation affects the growth and development of spinal development. In this study, we investigated the role of DNA methylation in CS. METHODS: The target region DNA methylation level in the peripheral blood of patients with CS was analyzed. Through in-depth analysis, genes closely related to the growth and development of the vertebra were identified. EdU staining was performed to verify the role of differentially expressed genes in chondrocyte proliferation. RESULTS: The hypermethylated KAT6B gene was observed in patients with CS, and was positively correlated with the Cobb angle. KAT6B was primarily expressed on chondrocytes. The promoter of KAT6B in CS patients was hypermethylated, and its expression was significantly reduced. Further mechanistic studies revealed that EZH2 mediated trimethylation of lysine 27 on histone H3 of the KAT6B promoter. Overexpression of KAT6B in CS-derived primary chondrocytes can significantly promote chondrocyte proliferation, which may be related to activation of the RUNX2/Wnt/ß-catenin signaling pathway. CONCLUSION: Epigenetic modification of KAT6B may be a cause of CS. If similar epigenetic modification abnormalities can be detected through maternal liquid biopsy screening, they may provide useful biomarkers for early screening and diagnosis of CS.

Dysregulation of the ghrelin/RANKL/OPG pathway in bone mass is related to AIS osteopenia.

BACKGROUND: Osteopenia has been well documented in adolescent idiopathic scoliosis (AIS), and ghrelin has been shown to have a positive effect on bone metabolism. However, the circulating level of ghrelin is increased in AIS osteopenia, and the relationship between ghrelin and low bone mass in AIS osteopenia remains unclear. METHOD: A total of 563 AIS and 281 age-matched controls were recruited for this study. Anthropometry and bone mass were measured in all participants. Plasma ghrelin levels were determined by enzyme-linked immunosorbent assay (ELISA) in both AIS and control groups. An improved multiplex ligation detection reaction was performed to analyze single-nucleotide polymorphisms (SNPs). Facet joints were collected and subjected to immunohistochemistry; osteogenic gene and protein expression was also measured. Furthermore, primary cells were extracted from facet joints and bone marrow to observe the response to ghrelin stimulation. RESULTS: The body mass index was lower and circulating ghrelin was markedly higher in the AIS osteopenia group than in the control group. No significant difference was observed in four ghrelin level-related SNPs between the AIS osteopenia and control groups. RNA and protein analyses revealed higher RANKL/OPG and lower runx2 levels in AIS cancellous bone. Compared with normal primary osteoblasts and BMSCs, AIS osteopenia primary cells were insensitive to the same ghrelin concentration gradient and showed lower osteogenic ability, increases in OPG and decreases in RANKL. CONCLUSION: Our results indicate that high circulating ghrelin levels may not result from gene variations in AIS osteopenia. Dysregulation of the ghrelin/RANKL/OPG pathway may lead to decreased osteogenic ability of osteoblasts and BMSCs, which may be related to lower bone mass in AIS osteopenia.

MicroRNA-30a regulates chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells through targeting Sox9.

Cartilage injury is difficult to repair since the cartilage tissue lacks self-restoration ability. Improved formation of chondrocytes differentiated from the mesenchymal stem cells (MSC) by genetic regulation is a potentially promising therapeutic option. SOX9 is a critical transcription factor for mesenchymal condensation prior to chondrogenesis. Previous studies demonstrated that several microRNAs (miRNAs or miRs) play a critical role in the chondrogenic differentiation of MSCs. However, the interactional relations between miR-30a and SOX9 during chondrogenic differentiation of MSCs need to be further elucidated. In the present study, human bone marrow-derived mesenchymal stem cells have been isolated and induced into chondrogenic differentiation to imitate the cartilage formation in vitro. Additionally, the expression levels of several miRNAs that were reported to interact with the SOX9 3'untranslated region (UTR) were examined by using reverse transcription-quantitative PCR. The interactional relations between candidate miRNAs and SOX9 were verified with the transfection of a miRNA mimic or inhibitor and a luciferase reporter gene assay. The results indicate that miR-30a and miR-195 were consistently increased during MSC chondrogenic differentiation. Additionally, the binding of miR-30a to the SOX9 3UTR was verified. Then, the authors upregulated the expression of miR-30a and found that MSC chondrogenic differentiation was inhibited. Taken together, the results of the present study demonstrate that miR-30a has a negative regulatory effect on MSC chondrogenic differentiation by targeting SOX9. Advances in epigenetic regulating methods will likely be the future of systemic treatment of cartilage injury.

Muscle Injury Associated Elevated Oxidative Stress and Abnormal Myogenesis in Patients with Idiopathic Scoliosis.

Idiopathic scoliosis (IS) is a disease with unknown etiology characterized by spinal rotation asymmetry. Reports describing the histochemical and pathological analyses of IS patients have shown that necrosis, fibrosis and fatty involution occurred on the apex paraspinal muscles. However, research on the changes in the paraspinal muscles of IS patients compared with those in matched controls is rare; thus, the basic mechanism of how paraspinal muscles are injured in IS patients is still unclear. In this study, we investigated the morphological changes of paraspinal muscles in the control group and IS patients, and the possible mechanisms were examined in vivo and in vitro. Increased myofiber necrosis was found on both sides of the apex paraspinal muscles of IS patients compared with those of the control group, and the number of TUNEL-positive apoptotic cells was also increased. Apoptosis signaling pathways, including pro-apoptosis proteins such as cleaved-caspase 3 and cytochrome c, were markedly upregulated, whereas the anti-apoptotic Bcl-2/Bax was significantly downregulated in IS patients compared with the control group. Moreover, PGC-1α and SOD1 were upregulated in accordance with the increased ROS production in IS patients. The distribution of myofiber types, as well as the mRNA levels of type IIa myofiber marker MYH2 and the important myogenesis regulator MYOG were remarkably changed in IS patients. In addition, C2C12 or human skeletal muscle mesenchymal progenitor cells treated with antimycin A in glucose-free and serum-free culture medium, which can activate oxidative stress and induce apoptosis, showed similar patterns of the changed distribution of myofiber types and downregulation of MYH2 and MYOG. Altogether, our study suggested that the extents of severe muscle injury and accumulated oxidative stress were increased in IS patients compared with the control group, and the abnormal myogenesis was also observed in IS patients. Since elevated oxidative stress can lead to apoptosis and the dysregulation of myogenesis in muscle cells, it may be associated with the pathological changes observed in IS patients and contribute to the development and progression of IS.

Changes in circulating cell-free nuclear DNA and mitochondrial DNA of patients with adolescent idiopathic scoliosis.

BACKGROUND: Adolescent idiopathic scoliosis (AIS) which characterized by complex three-dimensional deformity of spine has been difficult to cure because of the unknown etiopathology and uncertainty of progression. Nowadays, circulating cell-free (ccf) DNA was found to be a potential biomarker for several benign and malignant diseases. However, whether ccf DNA can be a biomarker for AIS has not been reported yet. In this study, we investigate the circulating cell-free nuclear DNA (ccf n-DNA) and mitochondrial DNA (ccf mt-DNA) concentrations in the plasma of patients with AIS and controls (CT), and the changed plasma ccf n-DNA and ccf mt-DNA levels and their association with clinical parameters were assessed. METHODS: The plasma of peripheral blood from 69 AIS patients and 21 age-matched CT was collected for ccf DNA analysis. Quantitative PCR was used to detect ccf n-DNA and ccf mt-DNA levels, and correlation analyses between the ccf n-DNA and ccf mt-DNA levels and clinical characteristics were conducted. Receiver operator curves (ROC) were used to analyze the sensitivity and specificity of ccf n-DNA and ccf mt-DNA levels to different characteristics. RESULTS: The plasma ccf n-DNA levels of both GAPDH and ACTB were significantly decreased in AIS patients compared with those in controls, while the plasma ccf mt-DNA levels did not changed. According to sex-related analyses, the ccf n-DNA levels in male CT-M was higher than that in female CT and male AIS, but the ccf n-DNA levels in female AIS was not significantly changed when compared with male AIS or female CT. However, the concentration of ccf mt-DNA in female AIS increased significantly when compared with male AIS. Surprisingly, Lenke type-related analyses suggested that Lenke type 1 patients had lower ccf n-DNA levels, whereas Lenke type 5 patients had higher ccf mt-DNA levels compared with those of controls. However, a lower sensitivity and specificity of AIS predicted by ccf n-DNA or ccf mt-DNA levels was observed, whether in total, by sex, or by Lenke type. CONCLUSION: Although with no/little predictive accuracy of AIS/progressed AIS by ccf DNA levels, significantly changed plasma ccf DNA levels were observed in AIS patients compared with those in controls.

Ghrelin up-regulates cartilage-specific genes via the ERK/STAT3 pathway in chondrocytes of patients with adolescent idiopathic scoliosis.

Adolescent idiopathic scoliosis (AIS) is a severe spinal deformity that often occurs during puberty. The occurrence of AIS is suggested to be related to abnormal development of cartilage. Our previous study found increased serum ghrelin levels in AIS patients that may linked to the development of AIS. However, whether ghrelin affects cartilage in AIS patients is unclear. We used quantitative real-time PCR (qRT-PCR) and immunohistochemistry to detect the expression of cartilage-specific genes and the ghrelin receptor, growth hormone secretagogue receptor (GHSR). The mRNA and protein levels of collagen II (COLII), SOX9, AGGRECAN (ACAN) and GHSR were higher in AIS patients than in controls. In addition, the protein levels of GHSR downstream signaling pathway members p-STAT3 (Ser727), and p-ERK1/2 were increased. Furthermore, we treated chondrocytes from AIS patients with 100 nM ghrelin, the cell proliferation assay and Western blotting showed that ghrelin promotes chondrocyte proliferation and enhances COLII, SOX9, ACAN, p-ERK1/2 and p-STAT3 expression, respectively. Interestingly, all these observed alterations were abolished by ghrelin + [D-Lys3]-GHRP-6 (a ghrelin receptor inhibitor) treatment. And after U0126 (an inhibitor of ERK1/2 phosphorylation) treatment, ERK1/2 and STAT3 (Ser727) phosphorylation was simultaneously suppressed indicating that ERK1/2 is an upstream pathway protein of STAT3 (Ser727). In conclusion, ghrelin plays an important role in upregulating cartilage-specific genes on AIS primary chondrocytes by activating ERK/STAT3 signaling pathway.

Suv39h1 promotes facet joint chondrocyte proliferation by targeting miR-15a/Bcl2 in idiopathic scoliosis patients.

BACKGROUND: Idiopathic scoliosis (IS) is a complex disease with an unclear etiology, and the worldwide prevalence is approximately 2-3%. As an important link between environmental factors and phenotypic differences, epigenetic changes, such as lncRNA, miRNA, and DNA methylation, have recently been reported to be associated with the development of IS. However, the correlation between histone methylation, another classical epigenetic mechanism, and IS has not been determined. In this study, we investigated the morphological changes, alterations in the levels of histone methylation, and cell proliferation-related pathway in inferior facet joint cartilage in 11 IS patients and 10 comparable controls. RESULTS: Compared with the control group, narrowed facet joint cartilage but increased proliferative chondrocytes and upregulated collagen type II (COL2A1) and B-cell lymphoma-2 (Bcl2) were observed in IS patients. Additionally, tri-methylation levels of H3K9 (H3K9me3) rather than other lysine sites were significantly increased in IS patients, coinciding with the upregulation of its specific enzyme, suppressor of variegation 3-9, drosophila homolog of 1 (SUV39H1). In addition, Bcl2-targeted miR-15a was downregulated in IS patients, and the level of H3K9me3 in the promoter region of the miR-15a host gene was remarkably increased in IS patients compared with the control group. Moreover, overexpressing SUV39H1 in ATDC5 cells with increased H3K9me3 levels led to similar changes, with increased expression of COL2A1 and Bcl2, decreased expression of miR-15a, and increased cell proliferation. CONCLUSIONS: Thus, our study suggests that increased chondrocyte proliferation occurs in the facet joint cartilage of IS patients compared with the control group and may be promoted by the elevated levels of H3K9me3 and SUV39H1, which regulate the miR-15a/Bcl2 pathway. This dysregulation of chondrocyte proliferation could result in abnormal spinal growth and may additionally participate in the development and progression of IS.

Adiponectin regulates bone mass in AIS osteopenia via RANKL/OPG and IL6 pathway.

BACKGROUND: Osteopenia have been well documented in adolescent idiopathic scoliosis (AIS). Adiponectin has been shown to be inversely proportional to body mass index and to affect bone metabolism. However, the circulating levels of adiponectin and the relationship between adiponectin and low bone mass in AIS remain unclear. METHODS: A total of 563 AIS and 281 age-matched controls were recruited for this study. Anthropometry and bone mass were measured in all participants. Plasma adiponectin levels were determined by enzyme-linked immunosorbent assay (ELISA) in the AIS and control groups. An improved multiplex ligation detection reaction was performed to study on single nucleotide polymorphism. Facet joints were collected and used to measure the microstructure, the expression of RANKL, OPG, osteoblast-related genes, inflammatory factors, adiponectin and its receptors by qPCR, western blotting and immunohistochemistry. Furthermore, primary cells were extracted from facet joints to observe the reaction after adiponectin stimulation. RESULTS: Compared with the controls, lower body mass index and a marked increase in circulating adiponectin were observed in AIS osteopenia (17.09 ± 1.09 kg/m2 and 21.63 ± 10.30 mg/L). A significant difference in the presence of rs7639352was detected in the AIS osteopenia, AIS normal bone mass and control groups. The T allele showed a significant higher proportion in AIS osteopenia than AIS normal bone mass and control groups (41.75% vs 31.3% vs 25.7%, p < 0.05). micro-CT demonstrated that the AIS convex side had a significant lower bone volume than concave side. RNA and protein analyses showed that in cancellous bone, higher RANKL/OPG and adipoR1 levels and lower runx2 levels were observed, and in cartilage, higher adipoR1 and IL6 levels were observed in AIS. Furthermore, convex side had higher RANKL/OPG, IL6 and adipoR1 than concave side. Compared with normal primary cells, convex side primary cells showed the most acute action, and concave side primary cells showed the second-most acute action when exposed under same adiponectin concentration gradient. CONCLUSION: Our results indicated that high circulating adiponectin levels may result from gene variations in AIS osteopenia. Adiponectin has a negative effect on bone metabolism, and this negative effect might be mediated by the ADR1-RANKL/OPG and ADR1-IL6 pathways.

Taxifolin Inhibits Receptor Activator of NF-κB Ligand-Induced Osteoclastogenesis of Human Bone Marrow-Derived Macrophages in vitro and Prevents Lipopolysaccharide-Induced Bone Loss in vivo.

It has been reported that taxifolin inhibit osteoclastogenesis in RAW264.7 cells. In our research, the inhibition effects of taxifolin on the osteoclastogenesis of human bone marrow-derived macrophages (BMMs) induced by receptor activator of NF-κB ligand (RANKL) as well as the protection effects in lipopolysaccharide-induced bone lysis mouse model have been demonstrated. In vitro, taxifolin inhibited RANKL-induced osteoclast differentiation of human BMMs without cytotoxicity. Moreover, taxifolin significantly suppressed RANKL-induced gene expression, including tartrate-resistant acid phosphatase, matrix metalloproteinase-9 nuclear factor of activated T cells 1 and cathepsin K, and F-actin ring formation. Further studies showed that taxifolin inhibit osteoclastogenesis via the suppression of the NF-κB signaling pathway. In vivo, taxifolin prevented bone loss in mouse calvarial osteolysis model. In conclusion, the results suggested that taxifolin has a therapeutic potential for osteoclastogenesis-related diseases such as osteoporosis, osteolysis, and rheumatoid arthritis.