| Objectives:The intervention of early-onset scoliosis(EOS)needs to allow the further development of spine and thorax while correcting the deformity.However,the current surgical correction surgery has certain shortcomings and limitations,such as large surgical trauma,early spinal fusion,internal fixation loosening and fracture and so on.It is particularly necessary to find or develop a new minimally invasive and less complication method to treat EOS.Based on the experimental basis that EOS was effectively induced in piglets by ablation of the vertebral growth plate with electrocoagulation of the vertebral growth plate at a previous stage,we performed microwave ablation(MWA)on one side of the vertebral body in piglets by using a combination of imaging,histology,and molecular Analysis to study:1)cartilage tissue changes and effects on neurological function after microwave ablation of the vertebral growth plate,to investigate the feasibility of partial microwave ablation of the vertebral growth plate to regulate spinal growth;2)different spinal segment numbers in piglets were ablated with different microwave ablation areas;to initially explored the regulatory ability of partial microwave ablation of the vertebral growth plate on spine growth regulation;and 3)determine and analyze the differential proteomics between the microwave ablated side and the non ablated side of the vertebral growth plate,to reveal the potential molecular mechanism of vertebral growth plate microwave ablation regulating spine growth panoramically.Methods:1.Feasibility of microwave ablation of the vertebral growth plate for spine growth regulation:26 piglets at 2 months of age were included,and were randomly assigned to 7 groups according to MWA powers:50 W(n=4),40 W(n=4),30 W(n=4),20 W(n=4),15 W(n=4),10 w(n=4)and sham operated(n=2)animals.One side of the L1-L3 vertebral growth plates in experimental groups wer ablated using different MWA powers.The sham operation group was only inserte the ablation needle for 30s without microwave ablation.The temperature changes o the posterior,anterior and lateral walls of the intervertebral disc and the center of the vertebral growth plate were monitored during ablation.The safe ablation time and energy were determined according to the temperature measured by the temperature probe around the intervertebral disc.The changes of animal neurologic function were observed after ablation.The spinal samples were obtained after the animal euthanasia and HE and TUNEL section staining of the vertebral growth plate was performed to evaluate the histological changes and cell necrosis of the vertebral growth plate,and to detect the ablation area.2.Feasibility of partial microwave ablation of vertebral growth plates fo regulating spine growth:based on the results of the previous study,the left vertebra growth plates in L1-L3 was ablated with 20W and 40s microwave energy.A total of 9 experimental pigs were included.At 2,4,and 6 weeks after the operation,three piglets were sacrificed,then spinal samples were obtained for HE,TUNEL staining and immunofluorescence of collagen I and II.The cartilage tissue changes and the effects on neurological function after microwave ablation of the vertebral growth plate were studied.The growth changes of the spine,vertebral body,and growth plate were observed.3.Preliminary study on the ability of partial microwave ablation of vertebra growth plate to regulate spinal growth:According to the numbers of segments and ablation needles,eighteen piglets were randomly divided into five groups:single segment with single needle group(n=4),single segment with double needles group(n=4),three segments with single needle group(n=4),three segments with double needles group(n=4)and a sham operation group(n=2).In the experimental group single or double needles with 20 W,40 s microwave energy were used to ablate one side of the vertebral growth plates on different segments(L1-L2 or L1-L4).Gross and radiographic examinations were performed at 1,2,and 3 months,then animals were sacrificed at 3 months after surgery for histologic examination of the vertebral growth plate.The relationship between ablation area,numbers of ablated segments and regulatory ability was investigated.In the sham group,three spine segment were punctured with double needles for 30s,and microwave ablation was not performed.4.Mechanism of partial microwave ablation of vertebral growth plate regulating spinal growth based on proteomics:Vertebral growth plate specimens from the ablated and non ablated sides at 3 months after microwave ablation were selected.The proteins between the two groups was detected by TMT proteomics technology.The up-regulated and down-regulated proteins were screened and identified according to the criteria that the absolute value of the difference multiple was greater than 1.2 times and the p value was less than 0.05.Bioinformatic databases were consulted to perform bioinformatic analysis on the function,localization,and signaling pathways of the differentially expressed proteins.The detected differential proteins,which were closely related to cell proliferation and apoptosis,were verified by qPCR.Results:1.Successful operations were achieved in all 35 piglets for the feasibility study of microwave ablation of vertebral growth plate to regulate spinal growth.Postoperative neurological functions were all grade 5 on the modified Tarlov scale.No signs of nerve root palsy or spinal cord injury were observed,as well as other serious complicationes were found.No cell necrosis was observed in HE staining of the spinal cord.2.Of the piglets in experimental stage I,the transverse diameter of the ablated L1-L2 intervertebral disc was 28.0±1.4 mm and the longitudinal diameter was approximately 17.8±1.6 mm.The transverse diameter of the ablated L2-L3 intervertebral disc was 28.5±1.9 mm and the longitudinal diameter was about 18.8±2.3 mm.No significant difference was found between the size of the L1-L2 and L2-L3 intervertebral discs(*P>0.05);thus,it can be deduced that there was no difference in the corresponding vertebral growth plate,and the differences in ablation energy due to differences in growth plate size was avoided.3.The safe MWA time at different microwave powers during microwave ablation of the vertebral growth plate in piglets was 17.0 ± 1.5 s at 50 W,23.0±2.3 s at 40 W,31.0± 3.1 s at 30 W,47.0 ±3.7 s at 20 W,70.0 ± 4.2 s at 15 W,and 158.0 ±5.0 s at 10 W.With power<15 W,the vertebral growth plate could not be effectively ablated within the safe ablation time.4.Microwave ablation of vertebral growth plate showed obvious boundaries on HE stained sections.Within the safe ablation times,the MWA size on hematoxylin and eosin slices on a transverse diameter was between 7 and 10 mm;and that on longitudinal diameter was mainly determined by the ablation needle length.5.Growth plates and annulus fibrosus growth on the ablated side were arrested at 2,4,and 6 weeks after microwave ablation of the vertebral growth plate,and the growth plate,vertebral body,showed progressive dysplasia,while the vertebral body showed obvious wedge-shaped changes and the spine also showed significan unbalanced growth.6.At 2,4 and 6 weeks after microwave ablation of vertebral growth plate TUNEL staining showed that the chondrocyte necrosis of vertebral growth plate or the ablation side was significantly higher than that on the non ablation side Immunofluorescence detection showed that the expression of type Ⅰ collagen on the vertebral growth plate of the ablation side was higher than that of the non ablatior side,and the expression of type Ⅱ collagen was lower than that of the non ablatior side.7.Progressive EOS in piglets successfully were induced by using 20 W(for 40 s to ablate different segments of the vertebral growth plates adjacent to L1-L4;single or double needles were employed(0.5 cm apart);no neurological impairment was found after operation.The Cobb angle of single segment ablation at 3 months after the operation ranged from 10 to 15°(mean,13°)and that of three segment ablations ranged from 12 to 20°(mean,17°);no statistically significant difference in Cobb angle between single needle groups and double needle groups.There was no statistically significant difference in Cobb angle between single needle group and double needle group.Some ablated spine segments showed bony fusion at 3 months after the ablation,and the bony fusion ratio was 30%.8.In the proteomic study of the microwave ablated versus non ablated side of the vertebral growth plateA total of 148 differentially expressed proteins were identified by screening criteria with the absolute value greater than 1.2 and the P value less than 0.05.Among them,53 of the up regulated proteins were identified,accounting for 36%of the total differential proteins.95 proteins were down regulated,accounting for 64%of the total differential proteins.Up-regulated proteins are mainly as follows:collagen triple helix repeat protein 1,72 kDa type Ⅳ collagenase,and glucosidase 2 subunit β Et al;Down regulated proteins are mainly as follows:collagenase in neutrophils,target protein of Nesh-SH3,keratocan protein and so on.According to KEGG signaling pathway analysis,the signaling pathways that changed after microwave ablation in vertebral growth plate were mainly enriched in Apelin signaling pathway,PI3K/Akt signaling pathway,apoptosis signaling pathway,necroptosis signaling pathway,autophagy,and iron death,and so on.9.Select the differential proteins and key proteins in the in the PI3K/Akt signaling pathway,apoptosis signaling pathway which is closely related to chondrocyte proliferation and apoptosis,the corresponding genes of the proteins were found,then verify the accuracy of the differential proteins and the activation state of the related pathways by RT qPCR analysis.The results demonstrated that the expression of the corresponding genes of the selected differential proteins was consistent with the results of proteomic analysis.Conclusions:1.This experiment is the first application of microwave ablation in cartilage thermal injury and vertebral growth plate ablation,which proves that vertebral growth plate ablation under limited microwave ablation energy is safe and feasible,and can effectively lead to the necrosis and apoptosis of vertebral growth plate chondrocytes.2.Partial microwave ablation of vertebral body can lead to growth retardation of the vertebral body and growth plate.It can effectively regulate the growth balance of spine and partially lead to local bone fusion of the spine.This ability is expected to be used to adjust the scoliosis deformity with growth potential,especially in the treatment of EOS.3.The proteins related to chondrocyte proliferation and death were differentially expressed in the vertebral growth plate after microwave ablation.It was verified at a molecular level that microwave ablation can obviously affect the proliferation and death of vertebral growth plate chondrocytes,thereby regulating spine growth.4.Partial microwave ablation of vertebral growth plate has a reliable ability to regulate spinal growth.Compared with other epiphyseal block techniques and open surgery,it has strong advantages such as minimally invasive(which can be completed by percutaneous puncture),less intervention segments(only the target segments),no internal fixation placement(economic and less complications).These advantages are of great significance for the treatment of EOS patients with weak physique and poor surgical tolerance. |
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