Expression And Role Of Wnt16B In Human Articular Chondrocytes

PartⅠ Isolation and Identification of Human Articular Chondrocytes in vitroObjective: To isolate and culture human articular cartilage cells and to observe the morphological characteristics of human cartilage cells.The study of the basic theory for the next step of artificial cartilage reconstruction and cartilage transplantation.Methods: Articular cartilage cells were obtained with IRB approval from femoral tissue discarded during hip replacement surgery.Human articular chondrocytes were isolated and culture by using three-step enzymatic digestion,and the cells were subcultured.The cells were identified by hematoxylin-eosin staining and Alician blue staining.Results: The cultivation of cartilage cells showed adhesion,deformation,polygonal or triangle after three days cultured.The cell colony increased,and intercellular start processes connected after cells were cultured for 6 days.About 9 days in culture,colony increased,adjacent cells connected with each other.The cells formed a cohesive multiplayer on plastic surface about two weeks.Hematoxylin-eosin staining showed nucleus stained purple,cartilage matrix stained red,and surrounding cells or cytoplasm stained amaranth.Alician blue staining showed that cell plasma and membrane presented with dark blue.The cells turned to dedifferentiation after six gennetations,and proligeration and growrh of chondrocytes was slowly.Conclusion: Experimental results demonstrated that,a system that can isolate and culture cartilage cell is successful established.PartⅡ Influence of Osteoarthritis Grade on Molecular Signature of Human CartilageObjective: Articular chondrocytes maintain cartilage matrix turnover and have the capacity for anabolic and catabolic activities that can be influenced by injury and disease.This study tested the hypothesis that catabolic genes are upregulated with regional osteoarthritis(OA)disease severity within a joint.Methods: With IRB approval,specimens of knee cartilage obtained as discarded tissues from subjects undergoing arthroplasty were partitioned for each subject by OA disease severity and evaluated for gene expression by RT-PCR.There was regional OA grade-associated upregulation of expected inflammatory mediators TNF-receptors,IFN-and Interleukins as well as genes encoding proteolytic enzymes,including Adamts-5 and MMPs.Osteoclast-related genes,cathepsin K,tartrate-resistant acid phosphatase(TRAP),RANKL,RANK,M-CSF and c-fms,but not Osteoprotegerin,were induced in advanced Grades.In vitro treatment of normal human chondrocytes with Interleukin-1β upregulated similar genes.Results: This study provides evidence that chondrocytes per se can be the source of osteoclast-related factors.Immunohistochemical staining showed that RANK-and RANKL-positive cells were abundant in advanced Grades,especially in chondrocyte clusters.This suggests a possible autocrine mechanism by which an osteoclast phenotype is induced in articular chondrocytes.Conclusion: In sum,these studies identified gene expression signatures in human OA cartilage based upon regional disease severity within a joint.There was an effect of OA Grade on expression of osteoclastic lytic enzymes and regulatory factors in human articular chondrocytes.Induction of an osteoclast-like phenotype in chondrocytes may be part of OA progression and suggests specific therapeutic approaches.Part Ⅲ Role for Wnt16 B in Human Articular Chondrocytes ProliferationObjective: To discuss the effect of Wnt16 B on human articular chondrocytes proliferation,and the role of coordination with the signal pathway.Methods: With IRB approval,specimens of knee cartilage obtained as discarded tissues from subjects undergoing total hip replacement for osteoarthritis,and evaluated for Wnt16 B m RNA expression by RT-PCR.We performed human articular chondrocytes proliferation assays to determine the growth rates of chondrocytes cocultivation with different concentrations of Wnt16B.To detect the number and activity of chondrocytes cocultivation with different concentrations of Wnt16 B by cell count and MTT assay.The different concentrations Wnt16 B protein was cocultivation with human articular chondrocytes.At 24 and 48 h after cocultivation with Wnt16 B protein,the expression of p53,p14,p16,p21,ATM,Cyclin-E,Cyclin-D1,SOX9,PCNA andβ-catenin expression were analyzed by RT-q PCR and Western blot techniques.To evaluate the non-Wnt pathway by which Wnt16 B activates Wnt pathway,we used small chemical molecule kinase inhibitors,including PI-3 kinase inhibitor LY294002,p38 MAPK inhibitor SB203580,p42/44 MAPK inhibitor PD098059,JNK inhibitor SP600125,PKC inhibitor Chelerythrine Chloride(CHE),PKA inhibitor H-89.Results:(1)With culture passage cells from P2 and P5,Wnt2 expression increased(1.0 times),and the expression of Wnt5 a was unchanged.Wnt16 B expression is reduced by 15%;(2)As shown by RT-PCR,human articular chondrocytes expressed Wnt16 B.With OA severity grade increasing,the expression of Wnt16 B in 5 cases chondrocytes was gradually increased,1.87 fold in grade 1 versus grade 0,1.8 fold in grade 2/3 versus grade 0.(3)0.1 ng/ml,1 ng/ml,10 ng/ml Wnt16 B and cartilage cells were cocultured for fifth days,the number of chondrocytes increased by 1.83,2.26,3.37 fold,and in cocultured for seventh days,increased by 1.32,1.59,2.13 fold respectively.(4)The p53 and p14 m RNA expression levels decreased with the dose of Wnt16 B increasing.In a dose-dependent manner,Wnt16 B upregulated Cyclin-E,Cyclin-D1,SOX9,PCNA and β-catenin gene expression but not P16,P21 and ATM gene expression.(5)As shown by Western blot,Wnt16 B upregulated Cyclin-D1 protein expression in a dose-dependent manner and increased nuclear β-catenin protein expression(2.1 fold versus control at 10ng/ml Wnt16B),while decreased p53 protein expression(0.75 fold versus control at 10ng/ml Wnt16B).There was dose-associated downregulation of p21 protein expression(0.32-,0.23-and 0.19-fold versus control respectively).There was little or no effect on PCNA protein expression in coculture with different dose Wnt16 B.(6)To examine the role of Wnt16 B in osteoblastogenesis,we studied the effects of Wnt16 B on ALP and osteogenic marker genes in human articular chondrocytes.ALP activities,ALP and RUNX2 gene expression of human articular chondrocytes in treated groups with different dose Wnt16 B were not modified versus control groups.(7)To evaluate the non-Wnt pathway mechanism by which Wnt16 B activates Wnt pathway,we used small chemical molecule kinase inhibitors.PI-3K inhibitor,p38 MAPK inhibitor,p42/44 MAPK inhibitor,and PKC inhibitor had no affects,JNK inhibitor(SP600125,20 m M)and PKA inhibitor H-89(30 m M)antagonized the stabilization of β-catenin by Wnt16 B in human articular chondrocytes.Conclusion:(1)Human articular chondrocytes expressed Wnt16 B and the expression of Wnt16 B was gradually increased with OA severity grade increasing.(2)Proliferation rate was significantly increased by co-cultures of Wnt16 B,indicating a positive role in cell growth and proliferation.(3)RT-PCR showed insignificant alteration of the cell-cycle gene expression in co-cultures with Wnt16 B cells compared with control cells,indicating that Wnt16 B may not be directly involved in the cell cycle to enhance cell proliferation.(4)Wnt16B did not stimulate osteoblastogenesis of human articular chondrocytes in vitro.(5)This result demonstrated that JNK and PKA pathways were required for stabilization of canonical Wnt intracellular molecule β-catenin by Wnt16 B in human articular chondrocytes.