Comparative Proteomics Study Of Chondrocytes Encapsulated In Alginate Under Dynamic Compression

Mechanical stresses are known to play important role on articular cartilage functions in vivo and also on cartilage explants and chondrocytes monolayer culture. However,the molecular events of chondrocytes in respose to dynamic stress are still not well understood so far.Differential proteomics can be applied to evaluate the significant proteins in cellular metabolism, which would aid in better understanding of the molecular mechanism of cartilage remolding under dynamic loading.Therefore, in the present study, we tried to set up an in vitro 3D loading model system similar to native joint cartilage and found out the major proteins involved in the chondrocytes cultured in alginate beads under dynamic stress loading by using comparative proteomics technology .Methods:1) rabbit chondrocytes were isolated from the articular cartilage of a 4-week-old rabbit by enzyme digestion method , cultured in DMEM supplemented with 10% FBS.the chondrocytes were identified with Toluidine blue and immunohistochemistry of collagen type II staining;2)P1 chondrocytes suspended in calcicum alginate beads were observed dynamically under phrase contrast microscope,the vability and proliferation of cells were evaluated by MTT,The quantity of GAG was determined by a modification of the dimethyl-methylene blue method,histological and immunohistochemistry staining were employed to evaluate cell morphology, matrix deposition and the presence of cartilage specific type II collagen. 3) alginate hydrogels ranging in stiffness were formed via varying the alginate solids concentration from 1% to 3%, The compressive modulus of each condition were characterized using INSTRON 3365,The articular chondrocyte were encapsulated in each hydrogel condition, and growth, morphology,and gene expression were assessed; 4) A dynamic compression unit was set up followed the method of Sharam,chondrocytes cultured in 2% alginate beads were exposed to 0.2 MPa,0.66Hz cyclic loadings for 4h,the total proteins extracted from either loading or unloadind cells were separated by two-dimensional gel eletrophoresis(2-DE).5)PMF was analyzed using Matrix assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS),the differentially expressed proteins involved in chondrocytes cultured in alginate beads under dynamic stress loading were identified with protein and pertide databases.Results:1) The chondrocytes obtained here showed favorable growth and function character. The phenotype expression of chondrocytes could be kept steadily for three passages. The growth curve which could reflect the proliferation ability of the cells was obtained.in vitro cultured chondrocytes could also experienced the same mature process as they growed in vivo and had the similiar biological activity as those cells in vivo. 2) chondrocytes cultured in alginate mantained the spherical morphology. The number of viability cells and glycosaminoglycan(GAG) production increased with time .Histological examination reveal chondrocytes retained their differentiation state in alginate beads.3) The contents of the alginate had a significant influence on compressive mechanical properties. 1% alginate conditions had significantly lower moduli(19.44±2.72) than the 2%(167.09±12.43) and 3%(226.24±19.82) alginate conditions. chondrocytes were proliferating more rapidly on lower stiffness gels(1%,2%) than on the higher stiffness gels(3%). After day 21, no further cell proliferation, but a slight reduction in cell number, especially, on the softest gel,the reduction is most obvious.Matrix accumulation also varied significantly with type of alginate used, At week 4, the 2% group resulted in the highest GAGs production , the expression of cartilage-specific genes, such as type II collagen and aggrecan ,in the middle stinffness alginate constructs showed the highest level expression;4)two-dimensional gel eletrophoresis showed significant differences between the dynamic stress group and unloading group. There are 1698±13 protein spots in the loading group and 1632±54 in the unloading group. 10 proteins were diferentially expressed by analyzing with soft ware compared with unloading group,among them, 2 proteins were new appearance, 2 proteins disappeared,6 proteins were up-regulated.5)Among the 10 diferentially expressed proteins,the PMFs of 8 proteins were obtained through MALDI-TOF-MS analysis.Fouthermore ,by searching in protein and pertide databases,six meaningfull proteins were identified,they were:prolyl 4-hydroxylase alpha I, pyruvate kinase,L-lactate dehydrogenase A,Prolyl 4-hydroxylase subunit beta,destrin isoform,alpha enolase. Conclusions :1) The method used in the present work for isolation and culture of chondocytes is simple and feasible.The chondrocytes cultured in vitro maintained the specific chondrocytes phenotype in the P0, P1, P2 passages,which is suitable for most experiments.2) Alginate induced chondrocytes cluster-like growth and increased the deposition of GAG as well as phenotype stability,which indicated that alginate is favored for chondrocytes growth.3) The physical properties of alginate gel influence the embedded cells bio-behave,alginate with the middle stiffness led to the highest GAG synthesize and the most stable phonotype maintaining.4) it's feasible to investigate the influence of dynamic compression on chondrocytes through the differential proteomics approach.5) Our research provided fundamental information on the differential expression of protein of chondrocytes cultured in alginate under dynamic stress. however, futher studies need to be done to better understand the molecular mechanisms of cartilage remodeling induced by dynamic stress loading.