低强度脉冲式超声对兔脱细胞脱钙骨基质负载关节软骨细胞及骨髓间充质干细胞复合体的影响

Effect of low-intensity pulsed ultrasound on cell-free demineralized bone matrix co-cultured with rabbit cartilage cells and bone marrow mesenchymal stem cells in vitro

背景：有关低强度脉冲式超声波促进关节软骨损伤修复的研究较多，可供选择的细胞支架亦较多，但是有关低强度脉冲式超声波的应用条件及构建出理想的组织工程软骨细胞支架尚未达到共识。目的：构建新西兰兔同种异体脱细胞脱钙骨基质负载关节软骨细胞及骨髓间充质干细胞的复合体，给予一定条件的低强度脉冲式超声波刺激，以探讨脱细胞脱钙骨基质作为组织工程软骨支架的可行性和低强度脉冲式超声波刺激对关节软骨细胞及骨髓间充质干细胞的促进作用。设计、时间及地点：多个样本观察，实验于2009-05/08在解放军第二军医大学生物医学工程研究所完成。材料：应用新型改良Urist法制备兔脱细胞脱钙骨基质。采用机械粉碎结合酶消化法获取兔关节软骨细胞；采用全骨髓漂洗法分离出骨髓间充质干细胞并加以纯化，进行体外单层培养扩增。方法：按与脱细胞脱钙骨基质复合的细胞成分不同及是否应用低强度脉冲式超声波刺激不同分为4组：软骨细胞组、骨髓间充质干细胞组、复合细胞组：脱细胞脱钙骨基质分别与软骨细胞、骨髓间充质干细胞、软骨细胞/骨髓间充质干细胞复合，未进行低强度脉冲式超声波刺激。超声组：将脱细胞脱钙骨基质与软骨细胞/骨髓间充质干细胞复合，第2天进行低强度脉冲式超声波刺激，刺激频率1.0 MHz、瞬时空间强度10 mW/cm2，20 min/d。主要观察指标：①平面培养第2代关节软骨细胞及骨髓间充质干细胞行免疫组织化学检测。②新型改良Urist法制备的脱细胞脱钙骨基质行苏木精-伊红染色。③于细胞-支架复合第21天行Ⅱ型胶原的免疫组织化学检测。结果：①平面培养第2代关节软骨细胞行Ⅱ型胶原免疫组织化学染色，可见细胞形态为多角形、星形、圆形，有伪足伸出，胞质内容丰富，胞浆明显呈棕黄色，胞核为圆形。②平面培养第2代骨髓间充质干细胞的CD34免疫组织化学染色呈阴性，CD44及CD105免疫组织化学染色呈阳性。③新型改良Urist法制备的兔脱细胞脱钙骨基质内未见明显的细胞样结构，空隙大小均匀。④复合第21天后，骨髓间充质干细胞组的Ⅱ型胶原免疫组织化学染色阴性，软骨细胞组的Ⅱ型胶原免疫组织化学染色弱阳性，复合细胞组的Ⅱ型胶原免疫组织化学染色阳性，超声组的Ⅱ型胶原免疫组织化学染色呈强阳性。结论：①第1-3代关节软骨细胞及骨髓间充质干细胞与原代细胞的生物学性能相似。②在脱细胞脱钙骨基质内，软骨细胞和骨髓间充质干细胞可以保持较高的增殖能力。③10 mW/cm2的低强度脉冲式超声波不影响骨髓间充质干细胞活性，并且能够加速其向关节软骨细胞分化，但未发现有明显促进细胞增殖的作用。

BACKGROUND： Using low-intensity pulsed ultrasound （LIPU） to promote the repair of articular cartilage injury is very common, and we also have more options to choose the cytoskeleton, but the application conditions of LIPU and the appropriate cytoskeleton have not reached any consensus yet. OBJECTIVE： To investigate the feasibility of establishing tissue-engineered cartilage by cell-free allograft demineralized bone matrix （CFDBM） co-cultured with rabbit cartilage cells and bone marrow mesenchymal stem cells （BMSCs） in vitro, and to investigate the effect of LIPU on the cells in CFDBM. DESIGN, TIME AND SETTING： Multiple sample observation was performed at the Institute of Biomedical Engineering, Second Military Medical University of Chinese PLA from May to August 2009. MATERIALS： The CFDBM was prepared as modified Urists method; the cartilage cells were obtained using mechanical disintegration and enzyme digestion; the BMSCs were separated using whole bone marrow rinsing method, purified, and amplified layer by layer. METHODS： As CFDBM with a composite of different cellular components, and whether applying LIPU stimulation, the samples were divided into four groups： chondrocyte group, BMSCs group, compound group （CFDBM was compounded with chondrocytes, BMSCs, and chondrocytes/BMSCs, respectively, without LIPU stimulation）, and LIPU group （CFDBM was compounded with chondrocytes/BMSCs, and then the samples were stimulated with LIPU on the second day, 1.0 MHz frequency, 10 mW/cm2 transient spatial intension, 20 min/d）. MAIN OUTCOME MEASURES： ① The 2nd-generation of cartilage cells and BMSCs were examined by immunohistochemical method; ② The CFDBM prepared as modified Urists method was examined as HE staining; ③ The samples of four groups were examined by collagen II immunohistochemical staining on the 21st day. RESULTS： ① The collagen II immunohistochemical staining of the second generation of the articular cartilage cells showed that the morphostructure was polygon, star or round, and pseudopodia extended, and the cells were rich in cytoplasm; the cytoplasm was brownish yellow, and the cell nuclear was round. ② The result of immunohistochemical staining of BMSCs showed that, CD34 was negative, CD44 and CD105 were positive. ③ In the center of CFDBM prepared as modified Urists method, there was no obvious cell-like structure and the gap size was uniform. ④ On the 21st day after combining CFDBM with cells, collagen II immunohistochemical staining demonstrated that BMSCs group was negative, chondrocyte group was weak positive, compound group was positive, and the LIPU group was strongly positive. CONCLUSION： ① Biological property of the 1st-3rd-passage chondrocytes and BMSCs was similar to primary-cultured cells. ② Both chondrocytes and BMSCs had a highly proliferative ability in CFDBM. ③ 10 mW/cm2 LIPU could not affect activity of BMSCs but could promote differentiation into articular cartilage cells, and it also could not promote cell proliferation.