Experimental Research Of β-catenin In Mechanism On Early Articular Cartilage Degeneration Of Model Of Developmental Dislocation Of The Hip

[Background and Objective]Developmental dislocation of the hip (DDH) is one of the most common diseases in pediatric orthopedics. Osteoarthritis(OA) is the most frequent long term complication. Compared with the normal population, DDH OA is of early age of onset and serious clinical feature and most of the patients have to receive total hip arthroplasty (THA). Our previous studies have ensured the degenerative changes of the articular cartilage in early stage of DDH. However, the molecular biology of mechanism on early articular cartilage degeneration and OA is still unclear. In recent years, a lot of studies showed that P-catenin and its Wnt signal pathway play crucial role in articular cartilage formation and degeneration. But function of β-catenin in DDH early articular cartilage degeneration has not been reported. The purpose of this study is to detect the changes of the expression of β-catenin and collagen, matrix metalloproteinase (MMPs) as well as aggrecanase that relation to early articular cartilage degeneration, to explore correlation between β-catenin and DDH early articular cartilage degeneration, and we tried to identified the early degeneration mechanism of the DDH articular cartilage, to provide theoretical evidence for early preventment to DDH OA and explore a new idea for further study of relation of DDH and OA.[Methods]Part Ⅰ. To establish animal model of DDH, and observe pathohistologic changes of acetabulum and femoral head.1. According to swadding position of newborn is prone to DDH, we modified swadding position on20newborn Wistar rats though extension fixation of bilateral hips and knees with medical tape for10days, and then we observed the hip specimen to confirm the model is successful.2. That rats were sacrificed at ages of2,4,6,8weeks repectively. The size of acetabulum and femoral heads were compared between experimental and control group. In order to ensure the shape change in DDH model hips. In the mean time, the data was analysed by SPSS16.0software.3. Using the staining of Safranin O/Fast green to observe the changes of chondrocytes and extracellular matrix (ECM) of articular cartilage samples. Part Ⅱ. Detected the expression of Collagen X, matrix metalloproteinases-13(MMP-13), ADAMTS-4/5and β-catenin in articular cartilage of DDH model.1. At different ages,20samples were obtained from part Ⅰ.4μm sections and RNA was obtained from the hip joint.2. Apply the technique of immunohistochemical staining to respectively quantitate the expression of Collagen X, MMP-13and β-catenin in DDH and control rat cartilage.3. Applied qRT-PCR to detect mRNA expression of Collagen X, MMP-13, ADAMTS-4/5and β-catenin in DDH and control rat cartilage. Data was analysed by SPSS16.0software.Part Ⅲ. Apply the technique of culture chondrocytes in vitro to activate expression of β-catenin. Chondrocytes degenerative changes by β-catenin were detected.1. Primary articular chondrocytes were cultured from8weeks old normal10rats. Cells were tested by expression of Collagen Ⅱ by immunofluorescence and toluidine blue staining. The expression of Collagen X, β-catenin and MMP-13were detected by immunofluorescence and qRT-PCR. The expression of ADAMTS-5was detected by qRT-PCR.2. Primary articular chondrocytes were treated by LiCl to activate the expression of β-catenin. The expression of β-catenin, Collagen X, MMP-13and ADAMTS-5were detected by previous assay. Cell apoptosis was detected by TUNEL staining.3. The results were analyzed using SPSS16.0software.[Results]Part Ⅰ. To establish animal model of DDH, observe pathohistologic changes of acetabulum and femoral head.1. The achievement ratio of establishing animal model of DDH achieved100%(20/20).2. Necropsy specimens of rats in DDH model showed thicking of the capsule, irregular flat and shallow of the acetabular surface, embedding of softtissue, as well as flat and undeveloped femoral head. The sizes of acetabulum and femoral head were observed in DDH model smaller than in control group. Especially after4weeks of age, the development of acetabulum and femoral head stopped. The acetbulum became smaller and poor containment with femoral head aggrevated.3. Cartilage degeneration was observed by Safranin O/Fast green:The amount of negatively charged proteoglycans was dramatically decreased and cleft formation versus control counterparts, especially after at4weeks age. The chondrocytes of superficial zone in DDH model were rounded, aggregated and formed clusters. Cartilage degenerative changes aggravated with age-dependent.Part Ⅱ. To detect the expression of Collagen X、MMP-13、ADAMTS-4/5andβ-catenin in articular cartilage of DDH at different ages.1. The expressions of Collagen X and MMP-13by immunohistochemistry were higher than control group, especially after4weeks of age. The pecent of positive cell numbers of immunohistochemical staining were higher than that of control group. There was significant difference between two groups after4weeks of age (P<0.01).2. Increased mRNA expression of Collagen X, MMP-13and ADAMTS-5was observed in DDH model:For Collagen X and MMP-13, there was higher mRNA expression in DDH model than in control group (P<0.01). For ADAMTS-5, we found higher expression only at8weeks of age. However, there was no significant difference between two groups for ADAMTS-4.3. β-catenin:In control group, immunohistological analysis revealed that β-catenin was most highly expression at2weeks of age. Thereafter, the expression level of β-catenin steadily decreased in an age-dependent manner. In DDH model, there was no decrease tendency for the expression of β-catenin at different ages. Parallel analysis of β-catenin mRNA expression, by qRT-PCR, indicated that gene transcription followed the same expression pattern as was observed by immunohistochemistry between two groups.Part Ⅲ. Apply the technique of culture chondrocytes in vitro to activate expression of β-catenin. We detected chondrocytes degenerative changes by β-catenin.1. By immuofluoescence, we detected higher expression of Collagen Ⅱ. The toluidine blue staining was used for further testing cultures.2. When primary cultured articular chondroctes from hips of8week old rats were exposed to LiCl in culture, P-catenin expression was significantly increased by immunofluescence and qRT-PCR.3. β-catenin lead to chondrocytes degeneration:Higher expression of Collagen X and MMP-13were observed in LiCl-treated chondrocytes by immunofluescence and qRT-PCR. LiCl-treated chondrocytes showed higher expression of ADAMTS-5by qRT-PCR. TUNEL staining showed higher positive cells in Licl-chondrocytes. There was significant difference between two groups (P <0.01).[Conclusions]1. Early articular cartilage in DDH model appears retard development and then degeneration at the ages of4weeks. Early abnormal cartilage development and degeneration may be pathologic basis of long term OA in DDH.2. At the early stage of DDH model, the expression of Collagen X, MMP-13and ADAMTS-5correlated to early degeneration of articular cartilage increased obviously. The possibilities of Collagen X, MMP-13and ADAMTS-5as biological parameters to reflect the degree of degeneration need further studies.3. In the early stages of cartilage development, β-catenin signaling is necessary for articular cartilage growth, while after functional maturation it leads to degeneration and osteoarthritic-like chondrocytes. There was continuous higher expression of (3-catenin in early DDH model which may be result in abnormal articular cartilage development and degeneration.4. At the early stage of DDH, abnormal cartilage development results in continulous higher expression of β-catenin, and then cause expression of collagen X, MMP-13, and ADAMTS-5and increase of articular chondrocytes apoptosis, which lead to articular cartilage degenerative changes, which may be one of mechanisms that DDH eventually lead to the onset of OA.