Study On Teratogenicity After Repairation The Immature Skull Defect And Mechanisms Of The Skull Development

Backgroud Skull defects are common in neurosurgical patients and the skull repair is a routin treatment for the skull defect with a diameter more than 3cm in adult patients.Because the brains of Children are still developing,it remains unknown whether the skull repair for the Children with skull defect limits the development of the brains.There is no publicly accepted instruction for the treatment for the skull defect in Childern.Objective This study intends to find out whether the skull and brain development of the immature animal model is limited by the metal material(titanium mesh),which is applied to repair the skull defect in the young animal model.Further more,the mechanism of the skull development of the young animal model is also be preliminarily explored.Hopelly,the study will provide some theoretical basis for the treatment of skull defects in children.Study Contents(1)The 1-month-old sheeps were selected as the experimental animals.They were subgrouped into the experimental group(A),the experimental group(B)and the control group(C).Group A was further subgrouped into A1 and A2 groups.One titanium meshe was fixed on the skull along the parasagittal suture with 4 titanium nails and 4 free titanium nails were fixed symetrically in the other side of the skull of the sheep in group A1.One titanium meshe was fixed on the skull crossing sagittal suture with 4 titanium nails and 4 free titanium nails were fixed in front of this meshe symmetrically crossing the sagittal suture of the skull in group A2.A skull defect of size of 4 cm × 4 cm was established in the sheep in group B and then it was repaired with the titanium mesh with 4 titanium nails.The sheep in group C did not receive any intervention without skull defect,titanium mesh or nails fixation or skull repair.The CT scan was performed on the sheeps monthly.The changes of both the relative distance between the nails on the skull and the size of the the cranial cavity and brain tissue in both of the A and B groups were recorded.The sheeps in A,B and C groups were killed 10 months later.The relative distance between the free titanium nails and the fixed ones on the skull at different time points in both A and B groups were comparatively measured.The characteristics of the animal biological behaviors,the volume of the cranial cavity,the weight of the brain tissue and the microstructure of the skull and brain tissue in the A,B and C groups were consecutively compared too.(2)The effect of miRNA-539 on the differentiation and development of osteoblasts was studied based on the MC3T3-E1 cell lines cultured in vitro.The relationship between miRNA-539 and DLx2 gene was also studied.Method(1)Forty sheeps of 1-month-old were divided into three groups: experimental group A1,experimental group A2,experimental B and control group C,respectively.The control group did not receive any intervention.The titanium mesh was fixed on the skull by 4 titanium nails on one side of sagittal suture in group A1,the other 4 titanium nails were fixed on the skull completely symmetrical to the 4 ones fixed with the titanium mesh.In group A2,the titanium mesh was fixed by 4 titanium nails perpendicular to and crossing the sagittal suture,and then four free titanium nails fixed in front of this titanium mesh without fixing to the mesh.The skull defect with a size of 4CM × 4CM was established and then repaired with titanium mesh with 4 titanium nails in group B.The distance between the titanium nails was measured and recorded based on the monthly CT scan.The characteristics of the animal biological behaviors of the sheeps in different groups were evaluated.After 10 months,the weight and the volume of brain and the head circumference were measured when the sheeps were killed.The skull and brain tissue specimens under the titanium mesh were observed by microscope.All the results were compared with the control group C.(2)The cultured MC3T3-E1 cells were divided into experimental group,also known as mineralization group(ascorbic acid and β-glycerophosphate induced),and control group.The abundance of miRNA-539 at different time points in the experimental and control groups was detected by real time-PCR.The abundance of m RNA and the level of protein of DLx2 gene were inspected with RT-PCR and Western Blotting respectively at different time points in the experimental and control groups.The miRNA-539 lentivirus and blank control lentivirus was transfected into MC3T3-E1 cells which was cultured in mineralization-induced medium for 21 days.The abundance of the m RNA levels of miRNA-539 and DLx2 was measured by RT-PCR.The level of DLx2-regulated protein was measured by Western Blot,the activity of alkaline phosphatase is detected,and the structure of mineralized matrix is showed by alizarin red staining.Result(1)In A1 group: The distance between 4 free titanium nails in the control side of the fixed titanium mesh increased significantly.However,the body weight,head circumference,brain tissue volume and weight was not statistically significant compared with the ones in the normal control group C.The growth of cranial cells was higher in suture than that of other parts.In A2 group: the distance between 4 free titanium nails in front of the fixed titanium mesh crossing the sagittal suture increased significantly.However,there was no deformity in the development of the cranial cavity.Between the sheeps in group A1,A2 and C,there were no significant differences(p>0.05)in the characteristics of the animal biological behaviors,the relative items of the head circumference,the body and brain weight and volume.The growth of skull cells in bone joints was more active under the microscope.(2)In B group: the area of the defect bone was reduced with the active new bone growth covering the defect area.However,the thickness of the new bone was thin and brittle without a clear structure inside the diplo.The titanium mesh was fixed tightly in the skull without any movement.When compared with the sheep in group C,the structure of the new bone slice of the skull was different between the sheeps in gropu B and C when observed under the microscope.However,there was no significant difference between the brain tissue slices of these two groups(p>0.05)(3)The abundance of miRNA-539 at different time points in the control group was not statistically different(P> 0.05).While the abundance of miRNA-539 in the experimental group was significantly lower than the ones in the control group(P <0.05).The m RNA abundance and protein expression of DLx2 gene were not changed in the control group in different time points,but they significantly increased in the experimental group when compared with the ones in the control group.The m RNA abundance and protein level of DLx2 gene were significantly decreased in MC3T3-E1 cells transfected with lentivirus-miRNA-539(P <0.05).When compared with the control group,the Alkaline phosphatase activity significantly decreased,with a significant reduced osteoblast differentiation and mineralization nodules in the experinmental group.Conclusion(1)Four titanium nails fixed on the titanium mesh of immature skull move slippery accompanying with the growth of the skull without limitation to the growth and development of both the skull and brain.The growth of the young skull mainly locates in the suture.(2)Repairing the undeveloped immature skull defects with titanium mesh does not limite the growth of the skull and brain tissue.The new bone growth in the skull defect area is obvious resulting in a decrease in the size of the defect area.However,the structural of the new bone are defective and may not be effective in protecting the brain tissue.(3)miRNA-539 inhibits osteoblast differentiation and mineralization by regulating DLx2 gene expression.