Experimental Study Of Asymmetric Growth Of The Spine Modulated With Microwave Ablation On Different Key Growth Sites Of Vertebral Body In Immature Porcine Under CT Guidance

Objective: The treatment of early onset scoliosis should maintain the growth of spine,thorax and the development of lung while delaying or controlling the progress of the deformity.At present,the non-fusion surgery with the growth rod technology as the mainstream in our country has achieved satisfactory results in the treatment of EOS.However,the treatment of EOS is still full of challenges due to its large trauma and a series of related complications such as the need for regular distraction,repeated anesthesia,spontaneous fusion,infection risk,high expenditure,etc.after the use of internal fixation.Therefore,it is very important to explore a minimally invasive treatment without internal fixation in the treatment of EOS.In this study,the different key growth sites of the vertebral body,the growth plates(GP),neurocentral cartilages(NCS)and GP combined with NCS(GP+NCS),in immature porcine were punctured under the guidance of CT respectively,and then microwave ablation was used to regulate the asymmetric growth of both sides of the spine.Through imaging,histology and molecular biology methods,the modulated ability and mechanism of GP and NCS on the asymmetric growth of the spine were clarified.The purpose of this study was to explore a minimally invasive strategy without internal fixation in the treatment of EOS in order to avoid the problems of large trauma in open surgery,repeated operations and related complications after the use of internal fixation,and finally provide a new thought and approach for the treatment of EOS clinically later.Methods: 1.Experimental study of spinal asymmetric growth with the modulation of GP by microwave ablation under CT-guided in immature porcine:Fifteen 6-week-old piglets were randomly divided into GP group,sham group and control group.The left GP of the vertebral body in the GP group was punctured under the guidance of CT scan and then ablated with microwave,while the sham group only placed the ablation needle for 30 s after puncturing the left GP,and the control group give no intervention and only as controlled observation.2.Experimental study of spinal asymmetric growth with the regulation of NCS by microwave ablation under CT-guided in immature porcine: 15 immature porcine were randomly divided into NCS group,sham group and control group.The left NCS midpoint was punctured under CT guidance and then microwave ablation was performed in the NCS group.In the sham group,the left NCS midpoint was punctured under CT-guided and the ablation needle was only placed for 30 s and then removed.In the control group,no intervention was given.3.Experimental study of spinal asymmetric growth with the modulation of different key growth sites of vertebral body by microwave ablation under CT-guided in immature porcine.Fifteen immature porcine were randomly divided into GP group,NCS group and GP+NCS group.The GP,NCS and GP+NCS on the left side of the vertebral body were ablated under CT guidance respectively.The movement of bilateral hind limbs,urination and defecation of the experimental animals were observed after the operation.The heights of the vertebral body on the left,middle,right,anterior and posterior were measured on the median coronal and sagittal plane at the 1st,2nd and 3rd months post-op after CT scanning.The length,width of pedicle on the left and right side and spinal canal area were measured paralleled to the pedicle plane.Three months after operation,the experimental animals were euthanized for gross observation and HE staining,TUNEL staining,ALP and SOX9 immunofluorescence staining to insight the histological changes,chondrocyte necrosis,osteogenesis and chondrogenesis characteristics of different key growth sites of the vertebral body after microwave ablation.4.Proteomic study on the modulation of asymmetric growth of spine by microwave ablation of key growth sites of vertebral body in immature porcine: Three months after ablation,the GP and NCS samples of the ablated side and contralateral vertebral bodies were taken from the GP+NCS group,and the amount of protein in the tissues of both sides was detected by TMT proteomics technique.The difference of up and down-regulation protein was identified by taking P<0.05 and multiple of difference>1.5 or <0.67.The function,location and signal pathway of differentially expressed proteins were analyzed after inquiring the bioinformatics database.The detected differentially expressed proteins were verified.Results: The operation was successfully completed in all the experimental animals.The activities of both hind limbs and urination and defecation were normal post operation.The punctured points healed well and no infection occurred in all the animals.1.In the experimental study of asymmetric growth of the spine modulated with microwave ablation on GP of vertebral body in immature porcine under CT guidance:Bilateral asymmetric growth of the spine occurred and scoliosis formed in GP group 1month post operation and the angle increased with the prolongation of observation time.Scoliosis was not observed both in the sham and control group.The Cobb angles on coronal plane in the GP,sham and control group were 18.98±2.87°,0.39±0.25°,0.32±0.25° and 22.91±2.9°,0.94±0.66°,1.02±0.95° and 27.78±1.73°,3.1±1.34°,2.63±1.74° at the 1st,2nd and 3rd month post-op respectively.The height of the left,middle and anterior of the ablation vertebral body in the GP group was significantly lower than that in the control and sham group,and the height on the ablated side of vertebral body in the GP group was significantly lower than that in the contralateral side,and the difference was statistically significant at each time point(P>0.05).Compared with the sham and control group,there was no difference in the height of the vertebral body on the right side,the height on the posterior,the length of pedicle on the left and right side and the area of the vertebral canal in the GP group.2.In the experimental study of asymmetric growth of the spine modulated with microwave ablation on NCS of vertebral body in immature porcine under CT guidance:Asymmetric growth on bilateral sides of the spine was seen and scoliosis deformity formed at 1 month after operation in the NCS group.The Cobb angle increased with the observation time.Scoliosis did not appear in both the sham and control group.The coronal angles of NCS,sham and control group were 13.46±1.62°,0.06±0.14°,1±2.24° and 15.99±2°,1.19±0.93°,0.46±0.43° and 19.63±3.22°,2.63±1.9°,1.58±1.94° respectively at the 1st,2nd and 3rd month post-op.At each time point,the height of the left and middle part of the ablation vertebral body,the length of the left pedicle and the area of the vertebral canal in the NCS group were significantly lower than those in the control and sham group.The height of vertebral body and the length of the pedicle on the ablation side in the NCS group were significantly lower than those of contralateral side,and the difference was statistically significant at each time point(P>0.05).Compared with the sham and control group,there was no difference in the height of vertebral body on the right,anterior and posterior side,the pedicle length on the right and the pedicle width on the left and right side in the NCS group.The rotation the ablation vertebral body occurred and rotated to the contralateral side on the horizontal plane.The rotation angle of the ablated vertebral body was 4.52±0.91°,6.39±0.95° and 9±1.49° respectively at 1,2 and 3 months after operation.Histological examination at 3 months post-op showed that part of the cartilage cells in the vertebral body of the ablation side of GP group and NCS group were partially filled with large number of fibrous connective tissue after coagulation necrosis,and plenty of fibroblasts were seen in the fibrous tissue,with a small amount of new bone formation around.Whereas cartilage cells in the vertebral body of the non-ablation side,Sham group and Control group were arranged normally and orderly.3.In the experimental study of asymmetric growth of the spine modulated with microwave ablation on different key growth sites of vertebral body in immature porcine under CT guidance: Asymmetric growth of the bilateral spine occurred in all the GP,NCS and GP+NCS group.Scoliosis deformity formed in 1 month after operation and the scoliosis progressed with time.The coronal angles of the GP,NCS and GP+NCS group were 18.54±3.09°,13.15±1.9°,28.01±1.78° and 22.3± 3.47°,15.31±1.71°,34.03± 2.89° and 27.45±2.38°,18.94±2.69°,38.47±2.89° respectively at the 1st,2nd and 3rd month post-op operation.At each time point,there was a significant difference in the left height of the ablation vertebral body in the three groups and GP+NCS group<GP group<NCS group.The left pedicle length and spinal canal area in the NCS and GP+NCS group were smaller than those in the GP group and the difference was statistically significant(P<0.05),but there was no significant difference between NCS and GP+NCS group(P>0.05).The anterior height of vertebral body in GP and GP+NCS group was lower than that in NCS group and the difference was statistically significant(P<0.05),but there was no significant difference between the GP and GP+NCS group(P>0.05).In the GP+NCS group,the height of the vertebral body and the length of the pedicle of vertebral arch on the ablation side were significantly shorter than those on the contralateral side,and the difference was statistically significant(P<0.05).Histological examination at 3 months post-op showed that most of the chondrocytes in the ablation side of the vertebral body in GP and NCS group were coagulated and necrotic,partially filled with a large amount of fibrous connective tissue.A large number of fibroblasts were seen in the fibrous tissue.A small amount of new bone formed around and a small amount of chondrocytes remained locally.The chondrocytes of the non-ablated side and the vertebral body of Sham and Control groups were arranged normally and orderly.4.Proteomic study on the modulation of asymmetric growth of spinal column by microwave ablation of key growth sites of vertebral body in immature porcine: A total of 65 differentially expressed proteins were identified by the standard of multiple difference>1.5 or <0.67 and P<0.05,of which 23 were significantly differentially expressed up-regulated proteins and 42 were significantly differentially expressed down-regulated proteins.Enrichment analysis revealed that they mainly located in ECM,plasma membrane,nucleus and cytoplasm,and may participate in the regulation of ECM,immunity,inflammation,biological force,vesicular transport,intercellular communication and other functions and signal pathways.COL11A1,THBS2,ASPN,COL9A2,POSTN and COL9A1 are central proteins in the PPI network of these differentially expressed proteins.We verified that the expression differences of COL9A1,COL9A2,PLA2G4 A,THBS2,CAV1 and CAV2 were consistent with the results of proteomic analysis.Conclusions: 1.Safe,accurate and minimally invasive puncture can be done and microwave ablation of different key growth sites of the vertebral body can be performed under the guidance of CT scan.The asymmetric growth of the bilateral spine can effectively be modulated.The ablation of the key growth sites of the vertebral body can regulate the asymmetric growth of the spine without internal fixation.The vertebral body on the ablated side grew slowly and the opposite side grows normally post-op,resulting in asymmetric growth of the bilateral sides of the spine,and eventually forming a deformity with scoliosis toward the non-operative side.2.Intervention of key growth sites on the straight spine by microwave ablation can regulate the asymmetric growth of both sides of the spine and induce scoliosis.According to the reverse principle,the intervention of the key growth sites on the convexity of the vertebral body in the patients with asymmetric growth of EOS can also regulate the asymmetric growth of the initial spine to halt or delay the progression of the deformity,and even gradually correct the deformity using the growth potential of the concavity.3.The asymmetric growth of the spine can be regulated by the ablation of GP or NCS on one side of vertebral body in immature porcine.The intervention of GP on one side mainly affected the growth of the anterior structure thus regulating the longitudinal asymmetric growth of the spine.Intervention of NCS on one side not only affected the posterior structures such as the pedicle and spinal canal,but also had a certain influence on the longitudinal growth of the anterior vertebral body,but its role on the longitudinal growth of the spine was less than that of GP.Therefore,GP and NCS are the key parts of spinal growth and play a certain role in the formation and progression of scoliosis.GP is mainly responsible for the longitudinal growth of the spine,while NCS contributes to the growth of part of the anterior structure in addition to the posterior structure of the vertebral body.4.Intervention on the ipsilateral NCS while ablation of one side of the vertebral body GP can produce synergistic effect and achieve the maximum growth modulation.5.Through differential proteomics research,we believe that microwave ablation may mediate the growth of key growth sites of the vertebral body by influencing the signal pathways such as ECM,PI3K-AKT,IHH,IL-17 and sulfite metabolic,thus forming scoliosis in immature porcine.