Experimental Research Of Effects Of β-catenin On The Proliferation And Apoptosis Of Chondrocytes During The Early Degeneration Of Dislocational Hip Of Rat

Background and objectiveDevelopmental dislocation of the hip is a common deformity in pediatric orthopaedics. Due to early screening, many of these patients undergo proper treatment and acquire prospective results. However, some unpleasant complications such as re-dislocation、 residual dysplasia of the hip occur, leading to the early osteoarthritis at adulthood and total hip arthroplasty inevitably, which aggravate the load of patients. Yet, the cellular and molecular mechanisms of early osteoarthritis resulted from DDH have been still unclear. Some studies reveal that P-catenin intervened wnt signaling pathway plays critical role in the development、proliferation、differentiation、apoptosis and stasis of inner environment. During our earlier studies, we found that P-catenin plays dural modification in the development and degradation of cartilage; meanwhile, β-catenin the mature chondrocytes activated by LiCl underwent the similar results as the patho physiological process of DDH. However, as well-known, the regulation of cellular activity of the chondrocytes like proliferation and apoptosis by β-catenin during the process of degeneration is still unknown. Therefore, we will apply the experimental model to simulate the national process in order to investigate the regulation of proliferation and apoptosis by β-catenin in process of degeneration, and provide a new therapeutic treatment with the relationship between DDH and OA.Methods60newborn Wistar rats were randomly divided into control group and experimental group (DDH group). The model was mimicked by swaddling position which was fixed with hip and knee extension by medical tap. The fixation lasted10days with tap replacement everyday for transient movement. After removal of the fixation, these rats were bred together. And the hips and their cartilage were executed at2-、4-、8-weeks age, the intact hips were cut from coronal plane after fixation and decalcification, and the photos were taken at fixed focus. Acetabular depth ratio (ADR) and femoral head ratio (FHR) were measured by IPP6.0. Then, the slices were made from these samples for Mankin score by safranin-O fast green and immunochemistry for β-catenin at series stage.Meanwhile, the chondrocytes at different stage were harvested for analysis of viability by CCK-8test and cell cycle、apoptosis by flow cytometry. At the same time, LiCl was added into normal chondrocytes of8-weeks in order to activate β-catenin. The same methods were repeated for the observation the changes of the proliferation and apoptosis as well as TUNEL immunofluorescence for cell apoptosis analysis. Every experiment in vitro was repeated three times. The data were represented with X±SD. student t-tests by SPSS16.0were applied for statistical analysis. P<0.05is considered to be significant.Results1、The gross sample and measurement28/30(ratio93.3%) rats were identified successfully by the gross sample. The hip didn’t get reduction after the removal of tap from the observation of gross sample, and the pathology of acetabulum、counterpart femoral head concomitant with capsule in DDH group was similar with the process of human DDH. However, in the4-week age, the acetabulum and femoral head was deeper and bigger than2-week age, and the femoral head deshaped seriously. However, it was still smaller than the corresponding control group. Till8-week age, the presentation of acetabulum and femoral head became shaped obviously, along with the thicken capsule. The relationship between acetabulum and femoral head mismatched seriously.2、Safranin O-fast green and immunochemistry stainingGradual depigment of safranin O concomitant with clustes、clefts and empty lacuna in the DDH group was found with age. The Mankin score of4-and8-weeks showed great significant difference, representing the process of early degeneration. In the normal group, P-catenin was expressed in the superficial and transitional zones; however, along with the adulthood coming, the expression of P-catenin became down-regulated swiftly. But it in DDH group still remained high expression along with grown-up.3、Identification of primary chondrocytesPosititive staining of Toluidine blue and collagen II was applied for identification of primary chondrocytes.4、Cellular activity of proliferation and apoptosisIn2-weeks, the proliferative ratio of experimental group (0.86) was15.1%faster than the control group (0.75); in4-weeks, the proliferative ratio of experimental group (1.14) was24.6%faster than that of control group (0.92); in8-weeks, the proliferative ratio of experimental group (0.78) was21.8%faster than that of control group (0.64).According to the flow cytometry, the proliferative index of experimental group(44.9%) was much higher than the control group(30.4%) in2-weeks; while it was38.4%in experimental group higher than33.7%in control group(P<0.05) in4-weeks; at8-weeks, it was26.4%in experimental group higher than22.4%in control group(P<0.05).Meanwhile, the apoptosis ratio of experimental group was55.1%, much higher than that of control group(22.1%P<0.01) at2-weeks; and no significant difference was present at4-weeks between two groups as experimental group(1.02%) and control group(0.91%); however, it was49.1%in experimental group, showing great difference compared to control group(0.75%P<0.01).5、The cellular activity after activation of β-catenin by LiClAfter activiation of LiCl, the expression of β-catenin by immunofluroence in cytoplasm and nucleus of normal chondrocytes of8-weeks significantly increased, especially in nucleus.The proliferative ratio after activiation of LiCl was0.58, decreased9%compared to that of normal chondrocyte (0.64) and25.6%significantly compared to that of chondrocytes fromThe proliferative index after activiation of LiCl decreased significantly from22.4%to18.5%(P<0.05), and was lower than that of experimental group of8-weeks (26.4%P<0.01).The apoptosis ratio was increased significantly from0.75%to33.1%(P<0.01); however, was much lower than that of experimental group of8-weeks (49.1%P<0.01). Conclusion1. After10days fixation of bilateral hips of neonatal rat, the model of developmental dislocation of hip was made successfully. However, the hip of model after removal fixation didn’t get reduction, and osteoarthritis-like pathologic changes along with development.2. In the process of degeneration of DDH, the chondrocytes showed increased proliferative activity and high apoptosis.3. Up-expression of β-catenin may cause inhibition of proliferation and increase of apoptosis, which may be one of the mechanisms of degeneration of DDH.