The Experimental Study Of The Effect Of Fluoride On The Susceptibility Of Developmental Dysplasia Of The Hip And Its Mechanism

Objective:Developmental hip dysplasia(DDH)is a common birth defect involving hips in children,which initially affects the gait of children at walking age and eventually develops into osteoarthritis.Therefore,it can seriously reduce the quality of life of children who are suffering.Early detection and prevention can greatly improve the therapeutic effect of DDH.However,the etiology of DDH is not clear,and it is generally accepted that it is caused by multifactor,such as genes and environment.Besides those susceptible genes,j oint laxity,intrauterine breech presentation and postnatal straight leg swaddling are also included as risk factors.Especially joint laxity is believed as the main risk factor.Thus,the increase of j oint laxity will significantly increase the susceptibility to DDH.Although it is devoid of convincing evidence for the correlation between DDH and trace element intake,more and more studies suggest that fluoride can also cause damage to the soft tissue,particularly to those rich in collagen.The joint capsule is a fundamental and important anatomical structure to maintain the mechanical strength and stability of the hip joint,and its component is collagen.Nonetheless,there are no reports on the pathogenesis of DDH caused by exposure to fluoride in previous studies,so this study established an animal model of fluorosis combined with DDH to determine whether excessive intake of fluoride can reduce the strength of hip joint capsule,then lead to joint laxity,increase the susceptibility to DDH and its molecular biological mechanism was further investigated.Methods:This experimental study is divided into three parts.Part ?:the effect of high fluoride exposure on the incidence of DDH in the early stage of life,Twelve 3-month-old Wistar pregnant mice were randomly divided into four groups:control group,50 mg/L fluoride group,100 mg/L fluoride group and 150 mg/L fluoride group.Pregnant rats were fed with water containing fluoride of same concentration after delivery until the young rats were weaned(21 days after birth).Immediately after birth,the infant rats were fixed in the straight legs swaddling position of their hind limbs for 4 days.The incidence of DDH in each group was observed.Part ?:the effect of fluoride on the morphology,structure and function of the hip joint capsule in normal rats and its mechanism.Nine 3-month-old Wistar pregnant mice(0 days of pregnancy)were randomly divided into three groups:control group,50 mg/L fluoride group and 100 mg/L fluoride group.Pregnant rats were still fed with water containing fluoride of same concentration after delivery until the young rats were weaned.The hip joint capsule of each group was collected,and the alignment of collagen fiber bundles on the surface of the joint capsule was observed by scanning electron microscope;the changes of collagen fiber diameter of joint capsule was observed by transmission electron microscope;and the changes of morphology and collagen fiber content of j oint capsule were observed by Masson staining.PCR and Western Blot were used to analyze the difference of mRNA and protein expression of Collal and Col3a1 in the hip capsule of each group.Apoptosis detection kit and Western Blot were utilized to detect the level of apoptosis and the expression of apoptosis-related proteins in the hip capsule of each group.Part ?:the effect of fluoride on collagen metabolism of rats' hip capsule primary fibroblasts and its mechanism.The primary fibroblasts were extracted from the hip joint capsule of normal rats by tissue block method;the toxic effect of fluoride on primary fibroblasts was observed by CCK-8 method in order to determine the intervention concentration of fluoride in the experimental group.The expression of Collal and Col3a1 in the experimental group and the control group was observed by cellular immunofluorescence;and the expression of apoptosis-related proteins in primary fibroblasts was determined by PCR and Western.The ultrastructure of primary fibroblasts was detected by transmission electron microscope.The proportion of apoptosis between the experimental group and the control group was analyzed by flow cytometry;and Mitochondrial Membrane Potential(MMP)was detected,moreover,the difference of oxidative stress between the experimental group and the control group was analyzed by detecting the content of reactive oxygen species(ROS),catalase(CAT),superoxide dismutase(SOD),total antioxidant capacity(AOC)and malondialdehyde(MDA).Results:Part ?:the effect of high fluoride exposure on the incidence of DDH in early life.1.The DDH model of chronic fluorosis was successfully established by high fluoride exposure in the early stage of life combined with straight leg swaddling after birth;2.Elevated fluoride exposure significantly increased the incidence of DDH caused by straight leg swaddling after birth.Part ?:the effect of fluoride on the morphology,structure and function of the hip joint capsule in normal rats and its mechanism.1.Without straight leg swaddling,there was no incidence of DDH merely by exposure to high fluoride in the early stage of life;2.The hip joint capsule of the fluoride group was thinner than that of the control group,the diameter of collagen fibers was slender,and the alignment of fiber bundles was loose and disordered;3.The mRNA level of Collal and Col3a1 in the joint capsule of the fluoride group was significantly lower than that of the control group,while the protein expression level of Collal showed a downward trend,which was opposite to that of Col3a1;4.The apoptosis of capsule cells in the fluoride group was significantly higher than that in the control group,and the expression level of apoptosis-related protein was also remarkably higher than that in the control group.Part ?:the effect of fluoride on collagen metabolism of primary rat articular capsule fibroblasts and its mechanism.1.The expression trend of Collal and Col3a1 in primary fibroblasts treated with fluoride was consistent with the experimental results in vivo;2.The mRNA level and protein expression level of apoptosis-related genes in fluoride-exposed group were significantly up-regulated in primary fibroblasts.3.The ultrastructure of primary fibroblasts was dramatically affected by exposure to fluoride,including mitochondria and endoplasmic reticulum;3.After exposure to fluoride,MMP of primary fibroblasts in fluoride group decreased,the production of ROS increased markedly,however,CAT,SOD and AOC didn't change significantly,additionally the content of MDA was higher than that in the control group.4.The proportion of early and late apoptosis in the primary fibroblasts exposed to fluoride was considerably higher than that in the control group.Conclusion:1.High fluoride exposure in early life will not directly lead to the pathogenesis of DDH,but will increase its susceptibility;2.High fluoride causes slender,sparse and disordered collagen fibers in the joint capsule,which leads to the decrease of mechanical strength and joint relaxation;3.High fluoride down-regulates the expression of Collal in the hip capsule,but up-regulates the expression of Col3a1;4.High fluoride activates oxidative stress of articular capsule fibroblasts,regulates mitochondrial endogenous pathway,and promotes apoptosis of articular capsule fibroblasts.