Experimental Research Of The Reversibility Of Cartilage Degeneration In The Acetabular Dysplasia

[Background]Acetabular dysplasia (AD), which may cause articular degeneration in the later stage, could lead to Osteoarthritis (OA) finally, which need total hip arthroplasty and bring great economic burden and physical sufferings to the patients. Therefore, it is need to be resolved urgently how to delay and avoid the OA in the treatment of the AD. However, the mechanism of the AD leading to the OA is still unclear now. In the past literature, the major pathological change of the OA is the cartilage degeneration, but there is only little study on the morphological and molecular changes of the AD. It hasn't been reported that if there is reversibility of the degeneration of the AD, nor when does it occur, nor to which extent it can reverse. In this study, serial immature rabbit AD models were established by adding mechanical change to the hip, and the mechanical change was removed for some models. The thickness, fibrosis, ultrastructure, typeⅠcollagen, typeⅡcollagen, integrinβ1 in the acetabular cartilage were observed and measured, in order to analyze the order and reversibility of the pathological changes of the acetabular cartilage, and to study the mechanism of the AD leading to the OA, and to present theory evidence for choosing the time point of treating AD in children.[Methods]1. Establishment, radiographic and morphological study of serial rabbit AD models1) Thirty 4 to 5-week-old New Zealand rabbits were randomly divided to three groups, namely A, B, C. And every group owned 10 rabbits. The left hind limb served as the experimental side, which was given cast immobilization, maintaining knee extended and hip flexed for 2,4,6 weeks for each group respectively. The right hind limb served as the control side with no treatment. Five rabbits were sacrificed from each group after achieving their own casting time, composing the immature group. And the other five were given cast removal and were sacrificed at 6 months old, composing the mature group.2) For immature rabbits, Posteroanterior pelvic radiograph was taken before casting and every 2 weeks after casting. For mature rabbits, the radiograph was taken before sacrifice. The acetabular index (AI), aectabular head index (AHI), and the Sharp's angle was measured on each radiograph.3) The whole articular cartilages of bilateral acetabula were removed from the layer of the subchondral bone. Each specimen was divided into three (iliac, pubic and sciatic) parts according to its anatomical region. The iliac part was used for the HE staining and the electron microscopy. The pubic and sciatic parts were used for the PCR and the Western-blot. Each specimen was fixed and stored in different ways according to the instructions of different experiment methods. After the HE staining using the routine method, the thickness and fibrosis of the cartilage were measured.4) The morphology of the collagen fibril in the surface of the cartilage was observed using scanning electron microscope. The necrosis and ultrastructure of the chondrocyte were observed by transmission electron microscope.2. Expression of the type I, II collagen and integrinβ1 in the acetabular cartilage of the AD1) Specimen treatment was explained in details in the first part.2) The mRNA expression of type I, II collagen and integrinβ1 were detected by the Real-time fluorescence quantitative Polymerase Chain Reaction (RTQ-PCR).3) The protein expression of type I collagen and integrinβ1 were detected by the Immunohistochemical staining (IHC).4) The protein expression of type I collagen and integrinβ1 were detected by the Western-blot.[Results]1. Establishment, radiographic and morphological study of the serial rabbit AD models1) Nine rabbits died during experiment, and 6 were added, so the survive rate was 75%. The final sample quality was 27. There were 5 rabbits in each immature group. There were 4,3,1 rabbits in mature group A, B, C respectively. The experimental side of the other 4 rabbits couldn't go back to the normal position in knee flexition and hip flexion after removing the cast, so they were considered as group D individually.2) In the immature group, the average AI value of the treated hip increased gradually after 2,4,6-week immobilization, and was higher than that before casting (p<0.05), and was higher than that of the control side in each time point (p<0.01). In the mature models, no significant difference in the AHI and the Sharp's angle was found between the two sides in group A and B. The sharp's angle on the experimental side was higher than the control side in group C, and the AHI was smaller. The same phenomenon existed in group D with statistical significance (p<0.01for the Sharp angle, p<0.05 for the AHI). And the sharp's angle on the experimental side in group D is also higher than that in group A (p<0.05).3) In the immature group, the thickness of the edge of the experimental acetabular cartilage was larger than the control side in group A (p=0.019), B (p=0.025), C (p<0.001). In the mature group, the thickness showed no significant difference between the experimental and the control side. Fibrosis was found in the experimental side, and there is 1,2,1,4 cases in group A, B, C, D respectively. In group D, statistical difference was shown compared to the control group (p=0.014).4) The collagen fibrils showed obvious gross in the articular surface of the cartilage on the experimental side in the immature group A, the slices were unsatisfactory in group B and C. As for the chondrocyte, obvious necrosis was found in the 2,3, 4 layer of the cartilage. The number of the necrosis was smaller in group B and C. In the mature group B, the chondrocyte was weakly stained, and the number of lysosome in the experimental side was much larger than the control side. The phenomenon was not found in group A.2. The expression of the typeⅠ,Ⅱcollagen and integrinβ1 in the acetabular cartilage of the AD1) TypeⅠcollagen:IHC showed expression of typeⅠcollagen in both sides. RTQ-PCR showed mRNA expression was higher in the mature group than the immature group, and was lower on the experimental side than the control side in group D (p<0.05).Western-blot showed the typeⅠcollagen protein expressed higher as the growth of the rabbit, and lower in the experimental side than the control side.2) TypeⅡcollagen:RTQ-PCR showed no significant statistical difference between the experimental and the control side. In the immature group, the mRNA expressed higher in group A, lower in group B, and higher in group C on the experimental side. And it expressed higher with the rabbit growth on the control side.3) Integrinβ1:IHC showed expression of the integrinβ1 in both sides. RTQ-PCR showed no statistical difference. Western-blot showed higher expression on the experimental side than the control side in mature group B.[Conclusions]1. Changes in morphology, radiograph, and ultrastructure were reversible in some stage in serial AD models, which might provide theory evidence for choosing the time point of treating AD in children.2. The edge of the acetabular cartilage became thicker in AD in immature rabbits, which occurred fibrosis in mature rabbits. This course and ultrasructure changes of the cartilage might be the pathological base from AD to OA.3. The results of the expression of TypeⅠandⅡin the acetabular cartilage in AD implied that they take part in the fibrosis of the cartilage.