Postoperative adding-on phenomenon in Lenke 1A/B and 2A/B adolescent idiopathic scoliosis: risk factors and predictive index.

PURPOSE: The aim of this study was to identify associated risk factors of distal adding-on phenomenon in Lenke 1A/B and 2A/B adolescent idiopathic scoliosis (AIS) patients and establish the corresponding prediction model. METHODS: The clinical data of 119 Lenke 1A/B and 2A/B AIS patients were retrospectively analyzed. Preoperative, first erect (FE) radiographic parameters and radiographic parameters at the last follow-up were measured. Patients were divided into the adding-on group and the no adding-on group according to whether the adding-on phenomenon was observed at the last follow-up. Univariate analysis and multivariate logistic regression analysis were used to establish the corresponding prediction model. RESULTS: Adding-on affected 39 (32.8%) patients at the last follow-up. Risser sign and 19 radiographic parameters showed significant differences between the two groups by univariate analysis. Stepwise logistic regression analysis found that the Risser sign and so on five predictor variable, and the nomogram was drawn. The calibration curve showed that the model fitted well. The area under the receiver operating characteristic (ROC) curve is 0.949. And the decision curve analysis curve model within the threshold range for interventions to improve clinical outcomes. There was no significant difference in SRS-22 scores between the two groups. CONCLUSIONS: This study established a prediction model with adding-on in Lenke 1A/B and 2A/B AIS patients. The nomogram contains five predictive variables, which can effectively predict the probability of adding-on phenomenon during follow-up, and may have greater clinical value for the treatment and prevention of adding-on phenomenon.

Analysis of risk factors associated with pre-myopia among primary school students in the Mianyang Science City.

Objectives To find out the prevalence rate of pre-myopia among primary school students in the Mianyang Science City Area, analyze its related risk factors, and thus provide a reference for local authorities to formulate policies on the prevention and control of myopia for primary school students. Methods From September to October 2021, Cluster sampling was adopted by our research group to obtain the vision levels of primary school students employing a diopter test in the Science City Area. In addition, questionnaires were distributed to help us find the risk factors associated with pre-myopia. Through the statistical analysis, we identify the main risk factors for pre-myopia and propose appropriate interventions. Results The prevalence rate of pre-myopia among primary school students in the Science City Area was 45.27% (1020/2253), of which 43.82% were boys and 46.92% were girls, with no statistically significant difference in the prevalence rate of myopia between boys and girls (2 =2.171, P=0.141). The results of the linear trend test showed that the prevalence rate of pre-myopia tends to decrease with increasing age (Z=296.521, P=0.000). Logistic regression analysis demonstrated that the main risk factors for pre-myopia were having at least one parent with myopia, spending less than 2 hours a day outdoors, using the eyes continuously for more than 1 hour, looking at electronic screens for more than 2 hours, and having an improper reading and writing posture. Conclusion The Science City Area has a high prevalence rate of pre-myopia among primary school students. It is proposed that students, schools, families, and local authorities work together to increase the time spent outdoors, reduce digital screens and develop scientific use of eye habits.

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.

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.

Halo traction combined with posterior-only approach correction for cervical kyphosis with Neurofibromatosis-1: minimum 2 years follow-up.

BACKGROUND: Surgical management of cervical kyphosis in patients with NF-1 is a challenging task. Presently, anterior-only (AO), posterior-only (PO) and combined anterior-posterior (AP) spinal fusion are common surgical strategies. However, the choice of surgical strategy and application of Halo traction remain controversial. Few studies have shown and recommended posterior-only approach for cervical kyphosis correction in patients with NF-1. The aim of this study is to evaluate the safety and the effectiveness of halo Traction combined with posterior-only approach correction for treatment of cervical kyphosis with NF-1. METHODS: Twenty-six patients with severe cervical kyphosis due to NF-1 were reviewed retrospectively between January 2010 and April 2018. All the cases underwent halo traction combined with posterior instrumentation and fusion surgery. Correction result, neurologic status and complications were analyzed. RESULTS: In this study, cervical kyphosis Cobb angle decreased from initial 61.3 ± 19.7 degrees to postoperative 10.6 ± 3.7 degrees (P<0.01), with total correction rate of 82.7%, which consist of 45.8% from halo traction and 36.9% from surgical correction. JOA scores were improved from preoperative 13.3 ± 1.6 to postoperative 16.2 ± 0.7 (P<0.01). Neurological status was also improved. There was no correction loss and the neurological status was stable in mean 43 months follow-up. Three patients experienced minor complications and one patient underwent a second surgery. CONCLUSION: Halo traction combined with PO approach surgery is safe and effective method for cervical kyphosis correction in patients with NF-1. A satisfied correction result, and successful bone fusion can be achieved via this procedure, even improvement of neurological deficits can also be obtained. Our study suggested that halo traction combined with PO approach surgery is another consideration for cervical kyphosis correction in patients with NF-1.

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.

Deformed Complex Vertebral Osteotomy Technique for Management of Severe Congenital Spinal Angular Kyphotic Deformity.

OBJECTIVES: To (i) introduce the deformed complex vertebral osteotomy (DCVO) technique for the treatment of severe congenital angular spinal kyphosis; (ii) evaluate the sagittal correction efficacy of the DCVO technique; and (iii) discuss the advantages and limitations of the DCVO technique. METHODS: Multiple malformed vertebrae were considered a malformed complex, and large-range and angle wedge osteotomy was performed within the complex using the DCVO technique. Patients with local kyphosis greater than 80° who were treated with DCVO and did not have tumors, infections, or a history of surgery were included. A retrospective case study was performed in these patients with severe angular kyphosis who underwent the DCVO technique from 2008 to 2016. Demographic data, the operating time, and the volume of intraoperative blood loss were collected. Spinopelvic parameters (pelvic incidence [PI], pelvic tilt [PT], and sacral slope [SS]), local and global sagittal parameters (deformity angle, thoracic kyphosis [TK], and lumbar lordosis [LL]), visual analog scale (VAS) score, and Oswestry disability index (ODI) score were recorded pre- and postoperatively. Paired t-tests (α = 0.05) were used for all data (to compare the mean preoperative value with the mean postoperative and most recent follow-up values). P < 0.05 was considered statistically significant. RESULTS: Twenty-nine patients with a mean age of 34 years (range, 15-55) were included in the final analysis. Seventeen patients were male, and 12 were female. The mean follow-up was 44 months (range, 26-62). The mean operating time was 299 min (range, 260-320 min). The mean blood loss was 2110 mL (range, 1500-2900 mL). Three patients had T7 -T8 deformities (3/29, 10.3%), six had T8 -T9 deformities (6/29, 20.7%), six had T9 -T10 deformities (6/29, 20.7%), 10 had T10 -T11 deformities (10/29, 34.5%), three had T11 -T12 deformities (3/29, 10.3%), and one had T9 -T11 deformities (1/29, 3.4%). The mean local deformity angle significantly improved from 94.9° ± 10.8° to 24.0° ± 2.3° through the DCVO technique, with no significant loss at the follow-up. Moreover, the global sagittal parameters and spinopelvic parameters exhibited ideal magnitudes of improvement; TK decreased from 86.1° ± 12.1° to 28.7° ± 2.5°, LL improved from 94.5° ± 4.1° to 46.1° ± 3.0°, and PI minus LL improved from -60.9° ± 6.5° to -13.7° ± 2.6°. Both the VAS and ODI scores significantly improved at the last follow-up. CSF fistula and neural injury did not occur during the perioperative period. At the last follow-up, fixation failure was not observed. CONCLUSION: The DCVO technique provides an alternative and effective method for the treatment of congenital severe angular spinal kyphotic deformities and may decrease the occurrence of perioperative complications.

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.

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.

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.

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.

Opposite Function of ERα and ERß in Controlling 17ß-Estradiol-mediated Osteogenesis in Osteoblasts.

Estrogen receptor plays critical roles in osteogenesis but the underlying mechanism remains unclear. In order to determine the effect of ERα and ERß on several critical factors in regulating osteogenesis in human osteoblasts. Cell based assy, RT-PCR and immunoblot analyses were used in the research. Both RT-PCR and immunoblot showed that gene expression of OPG, MBP2, TGF-ß, RUNX2, IGF-1 was significantly reduced while expression of RANKL was drastically increased after shRNA-based depletion of ERα in MG-63 osteoblasts. Surprisingly, 17ß-estradiol (E2) treatment led to remarkably reduced RANKL compared with that in E2 untreated cells. In contrast, ERß plays an opposite role in regulating gene expression of OPG, MBP2, TGF-ß, RUNX2, IGF-1 and RANKL. However, double depletion of ERα and ERß could not rescue the gene expression of these factors in vitro. Our results provide a novel mechanism of estrogen receptor in controlling osteogenesis in human cells as well as a potential clinic therapeutic target in human osteoporosis.