| Objective: We aimed to explore the association between the changes of chondrocyte cytoskeleton(CSK) and phenotype by modulating the chondrocyte CSK protein structure and measuring the alteration of chondrocyte chondrogenic phenotype.Methods: Chondrocytes from knee joints of New Zealand Rabbits were cultured as 3D micropellets for 14 days after isolation and in vitro 2D expansion. Chondrocytes randomly divided into control group,colchicine group, cytochalasin B group and acrylamide group.Colchicine(Colc), cytochalasin B(Cyto-B), or acrylamide(Acry) was added into culture medium to disrupt tubulin microtubule, actin microfilaments, or vimentin intermediate filaments respectively. After 7 days and 21 days of drug regulator, macroscopic observation, hematoxylin-eosin(H.E.) staining, Alcian blue staining, and immunohistochemistry(IHC) were done to analyze the changes of chondrocyte phenotype.Results: 1. After 14 days of pellet culture, chondrocye micropellet was observed as oval in shape, white in color, ~5 mm in size, elastic in stiffness.2. After 7 days of drug regulator, the quality of chondrocyte pellets in Acry-treated group was significantly different that in control, Colc-treated, and Cyto-B-treated groups( 2.8133?0.2444,4.97?0.0963, 4.7678?0.2997,4.84?0.324;P<0.05); while after 21 days of drug regulator, there was no significant difference in pellet morphology among all groups(8.0767?0.2779,8.8444?0.7012,8.4789?0.6473,8.4633?0.6837;P>0.05).3. H.E. staining results showed that at 7 days of drug regulator, the cells in Acry-treated group were less dense and homogeneous than cells in the other groups; at 21 days, chondrocytes formed cartilage lacuna phenomenon, cell distribution uniform among all groups.4.Alcian blue staining results showed that at 7 days of drug regulator, the level of Glycosaminoglycan in Acry-treated group was lower than the other groups( 0.1517?0.0484, 0.6012?0.0672, 0.5657?0.0514, 0.5686?0.0568;P<0.05); at 21 days, the level of Glycosaminoglycan was similar in all groups(1.6947?0.4163,1.5416?0.3437,1.7884?0.2861, 1.7382?0.3107;P>0.05).5. IHC results showed that at 7 days of drug regulator, Control,Colc-treated and Cyto-B-treated pellets expressed more type II collagen(0.0894?0.0054,0.0859?0.0024,0.0829?0.0066;P>0.05) and less type I collagen(0.0013?0.0002,0.0017?0.0002,0.0016?0.0056;P>0.05), while Acry-treated pellets expressed less type II collagen(0.0289?0.0099,0.0894?0.0054;P<0.05) and more type I collagen(0.1071?0.0012, 0.0013?0.0002;P<0.05); at 21 days, the expression of type I collagen( 0.0675?0.0056, 0.0693?0.0079, 0.0722?0.0051, 0.0746?0.0135;P>0.05) and type II collagen(0.1904?0.0471, 0.1904?0.0471,0.1856?0.0224,0.1772?0.0353;P>0.05)had no significant difference among all groups.Conclusion:1.Disruption of microtubules and actin microfilaments d idn’t change the level of GAG and COL II.2.Disruption of vimentin intermediate filaments resulted in down-reg ulation of GAG and COL II, and up-regulation of COL-I in short time, w hile prolongation of time without increasing the effect of strength, the expression of GAG and COL II was not influenced. |
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