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目的运用微管吸吮技术对海藻酸钠培养的兔膝关节软骨细胞力学特性进行定量分析,为软骨组织工程中相关力学因素的研究提供相应的技术参数并为最终利用组织工程治疗软骨损伤提供相关理论依据。方法2月龄新西兰白兔6只,无菌条件下分离其双膝关节,以培养液配制的0.4%Protease酶、0.025%Ⅱ型胶原酶顺序消化兔软骨分离细胞,以4×106/ml浓度与海藻酸钠混合,通过硅胶盘模制成圆形柱状细胞盘(25μl/个),CaCl2溶液中凝胶化5min,DMEM/F12无血清培养基加20%FBS(fatalbloodsolution)于6孔培养板中培养。于1、2、4周取出细胞盘,采用微管吸吮技术结合半无限体模型分析软骨细胞的力学特性(瞬时模量E0、平衡模量E∞、表观黏性μ)。利用流式细胞仪测定1、2、4周软骨细胞的凋亡。结果1、2、4周软骨细胞两两相比软骨细胞的早期凋亡和活细胞比较均没有统计学意义(P>0.05)。4周培养的软骨细胞黏弹性明显低于1周和2周(P<0.05),1周与2周之间比较不具有统计学意义(P>0.05);1、2、4周软骨细胞表现均为典型的黏弹性固体蠕变特征。结论通过微管吸吮力学测量海藻酸钠立体培养软骨细胞的力学特性相对较稳定,适合作为构建组织工程化软骨的种子细胞。
Objective To quantitatively analyze the mechanical properties of rabbit knee chondrocytes cultured in sodium alginate using microtubule sucking technique and to provide relevant technical parameters for the study of relevant mechanical factors in cartilage tissue engineering and to provide relevant theories for the ultimate use of tissue engineering in the treatment of cartilage damage in accordance with. Methods Six New Zealand white rabbits (2 months old) were randomly divided into four groups. The double knee joints were isolated under sterile conditions. The 0.4% Protease enzyme and 0.025% collagenase Ⅱ were used to digest rabbit cartilage. Mixed with sodium alginate and molded into a circular columnar cell plate (25μl / plate) through a silica gel plate. The plate was gelatinized in a CaCl2 solution for 5min. DMEM / F12 serum-free medium was added with 20% FBS (fatal blood solution) In training. Cell trays were removed at 1, 2, and 4 weeks. The mechanical properties of cartilage cells (transient modulus E0, equilibrium modulus E∞, apparent viscosity μ) were analyzed by microtubule sucking technique and semi-infinite body model. Chondrocytes apoptosis was measured by flow cytometry at 1, 2 and 4 weeks. Results There were no significant differences in chondrocytes apoptosis between the two groups at 1, 2, 4 weeks compared with chondrocytes (P> 0.05). The chondrocytes cultured for 4 weeks showed significantly lower viscoelasticity than those of 1 week and 2 weeks (P <0.05), and showed no significant difference between 1 week and 2 weeks (P> 0.05) All are typical viscoelastic solid creep characteristics. Conclusions The mechanical properties of chondrocytes cultured in vitro by microtubule sucking mechanics are relatively stable and suitable for constructing seed cells of tissue-engineered cartilage.