Etiological Analysis On Adolescent Idiopathic Scoliosis Using Single Cell RNA Sequencing And Related Clinical Studies Of Spinal Deformity

Chapter ? High throughput single-cell RNA Sequencing of cancellous bone tissue in adolescent idiopathic scoliosisPurposes:Adolescent idiopathic scoliosis(AIS)is the most common spine deformity,whereas the etiology remains unclear.Abnormal vertebral growth and development have been found in AIS patients,indicating that the proliferation and differentiation of bone development-related cells might play important roles in the pathogenesis.However,a comprehensive single-cell-level differentiation roadmap in AIS has not been achieved.Methods:The present study compared the single-cell level cellular landscapes between AIS patients and healthy subjects using high throughput single-cell RNA sequencing(sc RNA-seq),which covers multiple cellular lineages including osteoblast,chondrocyte,osteoclast and related immunocytes.We constructed the differentiation trajectories of bone development-related cell lineages through pseudo-time analysis,and the intercellularcommunication networks between bone development-related cells and immunocytes were further developed to study the potential impact of intercellular interactions on AIS pathogenesis.Results: A Total of 19 distinct cell subtypes are identified.Based on the comparisons of differentiation trajectories and intercellular-communication between AIS patients and healthy subjects,we accordingly speculated about some possible etiologies for AIS: a)Differentiation disruption of MSC-IGFBP5 in AIS may result in the loss of osteoblasts and dysfunctions of vertebral mineralization.The interactions between MSC-IGFBP and immunocytes and consequent abnormally activated signaling pathways might underlie the potential immunological pathogenesis of AIS.b)Chondrocyte differentiation could be blocked in AIS because of a prolonged HIF-1? signaling pathway,leading to an intracellular energy deficit.Furthermore,the loss of chondrocytes might give rise to defects of endochondral ossification in AIS vertebrae.Conclusions: Our single-cell analysis firstly reveals differences existed in the cellularstates between AIS patients and healthy subjects,and correspondingly provides the possible pathogenesis of osteoblast and chondrocyte differentiation dysfunction in AIS,which offering new insights into the etiology of AIS and potential targets for treatment.Chapter ? The effect of Rho A/ROCK signaling pathway abnormality on the differentiation of MSCs into chondrocytes in adolescent idiopathic scoliosis and its mechanismPurposes : Adolescent idiopathic scoliosis is one of the most common spinal deformities with unknown pathogenesis.The abnormal growth and development of skeletal cells has been concerned in the etiology of AIS.Our previous study also found that the differentiation of mesenchymal stem cells from AIS patients into chondrocytes was blocked.And the comparative analysis of intracellular signaling pathway suggested that there was an abnormal activity of Rho A/ ROCK signaling pathway in the mesenchymal stem cells of AIS patients,however,the present studies on Rho A / ROCK pathway in the mesenchymal stem cells in AIS are scarce.In this study,we intend to systematically study the regulatory effect and molecular mechanism of Rho A / ROCK signaling pathway abnormalities on the differentiation of mesenchymal stem cells into chondrocytes in AIS from gene,cell and protein levels.Methods: 30 Lenke 1 female AIS patients(AIS group)and 30 non scoliosis patients(control group)accepted surgery from January 2016 to June 2017 were selected.Single cell transcriptome sequencing was used to construct the differentiation trajectories of chondrocytes,and the difference of intracellular signal pathway between AIS mesenchymal stem cells and control group was analyzed.Western blotting was used to detect the expression of key proteins in Rho A/ROCK pathway between AIS and control group.Si RNA interference,adenovirus transfection and inhibitors were used to change the activity of Rho A/ROCK signaling pathway in mesenchymal stem cells.Chondrocyte proliferation and immunofluorescence staining,differentiation marker protein expression,downstream signal pathway key protein and phosphorylation level change were compared between AIS and control group at different timepoint.Results: SC RNA-seq showed that Rho A/ROCK signaling pathway was abnormally active in AIS MSCs,and Rho A protein expression was enhanced in nearly 50% of AIS patients by Western blotting,while the results of Alcian blue staining showed that differentiation of chondrocyte was decreased.MSCs were induced and cultured in vitro and compared at different timepoints among AIS H-Rho A group,control group,Rho A interference group,Rho A overexpression group and ROCK inhibitor group The phosphorylation level of LIMK,cofilin and MLC in Rho A/ROCK signaling pathway in AIS H-Rho A group was higher than that in the control group,suggesting that Rho A/ROCK signaling pathway in AIS MSCs was abnormally active,and the intensity of toluidine blue staining,collagen ? and F-actin immunofluorescence staining in chondrocytes was weaker than that in the control group,suggesting that the differentiation into chondrocytes was reduced;When interfering Rho A or inhibiting ROCK,the phosphorylation level of LIMK,cofilin and MLC was partially reduced,and the intensity of toluidine blue staining,collagen ? and F-actin immunofluorescence staining was increased;the overexpression of Rho A protein in MSCs of the control group could improve the phosphorylation level of LIMK,cofilin and MLC and reduce the differentiation into chondrocytes.Conclusions: the overexpression of Rho A in AIS MSCs may be the initiating factor of the abnormal activation of Rho A/ROCK signaling pathway.By increasing the phosphorylation level of downstream LIMK,cofilin and MLC,actin polymerization and cytoskeleton reorganization could be promoted.The abnormal activation of Rho A/ROCK signaling pathway in AIS MSCs can play an inhibitory role in the differentiation into chondrocytes.Therefore,it can constitute the etiology of differentiation disorder in AIS MSCs,and further lead to the abnormal growth and development of spine bon to participate in the pathogenesis of AIS.Chapter III Clinical studies on adolescent idiopathic scoliosisSection Postoperative shoulder imbalance in adolescent idiopathic scoliosis: risk factors and predictive indexPurposes: Postoperative shoulder imbalance(PSI)is a common complication in AIS.However,results of risk factors for PSI are conflicting.The objective of this study is to explore the risk factors associated with PSI in AIS,and to detect whether PSI could be predicted after correction surgery.Methods: Medical records of AIS patients who received correction surgery from January 2012 to January 2015 were reviewed.Antero-posterior films were evaluated before and after surgery,and at two years' follow-up.Patients were divided into two groups according to whether PSI occurred at the 2 years' follow-up.Risk factors of PSI were analyzed,and PSI Index was proposed and verified.Results: A total of 114 AIS patients(PSI/Non-PSI: 60/54)were included.Univariate analysis showed that preoperative PTC,preoperative PTC:MTC ratio,preoperative bending Cobb of PTC,preoperative bending Cobb of LC,postoperative PTC,postoperative AVT of PTC,AVT of PTC at follow-up,and Adding-on angle were significantly different between two groups.Adjusted logistic regression analysis showed that postoperative AVT of PTC and Adding-on angle were the primary contributors to PSI in AIS patients.PSI Index was defined as 1.2*Postoperative AVT of PTC+1.1*Adding-on angle.According to ROC curve,the cutoff point for PSI Index in predicting occurrence of PSI was 15.The positive predictive value and negative predictive value was 87% and 80%,respectively.Conclusions: T Postoperative AVT of PTC and Adding-on angle were the primary contributors to PSI.Adding-on and PSI are compensatory mechanisms for each other.PSI Index could be used to predict PSI effectively.To prevent the occurrence of PSI,we recommend sufficient correction of AVT of PTC,and Adding-on should be prevented as well.Section Postoperative neck tilt in Lenke 1 and 2 AIS patients after correction surgery: a novel predictive indexPurposes: Postoperative neck tilt(PNT)is a phenomenon in AIS patients that is distinct from shoulder imbalance.No studies have been performed to explore the risk factors for PNT in Lenke 1 and 2 AIS patients after correction surgery,and whether it can be predicted after surgery remains unknown.The objective of this study is to explore the risk factors for postoperative neck tilt(PNT)and introduce an index for prediction of PNT in Lenke 1 and 2 AIS patients after correction surgery.Methods: Medical records of Lenke 1 and 2 AIS patients who received correction surgery were reviewed from February 2013 to February 2015.Antero-posterior films were evaluated before and after surgery and at two years' follow-up.Patients were divided into two groups according to whether PNT occurred at the 2 years' follow-up.Risk factors of PNT were analyzed,and PNT Index was proposed and verified.Results: 102 Lenke 1 and 2 AIS patients were recruited in this study.According to the postoperative CAT(Cervical Axis Tilt),patients were divided into two group: PNT group(n=41,CAT?5°)and non-PNT group(CAT<5°,n=61).Postoperative T1 tilt,preoperative PTC(proximal thoracic curve),postoperative PTC and postoperative CB(coronal balance)were significantly different between two groups.Logistic regression showed that postoperative PTC and postoperative CB were the primary risk factors for PNT,which could be predicted by the regression equation: PNT Index=1.1* postoperative PTC-0.9*postoperative CB.On the basis of ROC curve,if PNT Index was more than 10,the occurrence rate of PNT was 86%.On the contrary,the rate of no PNT phenomenon was 81%.Conclusions: The prevalence of PNT in AIS patients after correction was 40.2%.PNT group and non-PNT group showed significant differences in postoperative T1 tilt,preoperative PTC,postoperative PTC and postoperative CB.Postoperative PTC and postoperative CB were the important factors for PNT in Lenke 1 and 2 AIS patients.PNT Index can be used to predict the occurrence of PNT effectively.