可注射性材料藻酸钠体外培养幼猪关节软骨细胞及形成组织工程化软骨的能力

In vitro culture of young swine chondrocytes in alginate gels to construct tissue-engineered cartilage

目的:观察以藻酸钠为载体体外培养幼猪关节软骨细胞的增殖及形成工程化软骨的能力;评价藻酸钠凝胶作为培养软骨细胞载体的可行性。方法:实验于2003-10/2004-03在中山大学附属第二医院实验中心完成。取3个月龄小型猪膝关节软骨,酶消化法得到高纯度软骨细胞,体外培养3代后与藻酸钠混合,调节软骨细胞密度为5×109L-1,接种于96孔培养板中,每孔接种50μL,25g/L氯化钙溶液凝胶化10min后,DMEM培养基加体积分数为0.1的胎牛血清于96孔培养板中培养。4周后取材观察,行苏木精-伊红染色、Masson染色及免疫组织化学分析,观察软骨细胞形态,Ⅱ型胶原表达及成软骨能力。结果:①单层培养的软骨细胞呈多角形,细胞浆内表达Ⅱ型胶原;藻酸钠与软骨细胞复合后,藻酸钠凝胶材料与透明软骨细胞之间嵌合好。②苏木精-伊红染色见幼猪软骨细胞在海藻酸钠中增殖成株状或岛状;Masson染色见类似软骨陷窝之间胶原被染成绿色,可见细胞周围胶原分泌;免疫组织化学染色见细胞岛及其周围基质呈橘红色,细胞分泌Ⅱ型胶原较多。结论:幼猪软骨细胞在藻酸钠中可良好生长增殖,幼猪软骨细胞和藻酸钠复合可在体外成功构建组织工程化软骨。

AIM： To observe the proliferation of chondrocytes from young swine cultured in vitro in alginate gels and the ability of tissue-engineered cartilage, so as to explorethe feasibility to culture chondrocytes with alginate gel as the carder. METHODS： The experiment was carded out in the Experiment Center of Second Affiliated Hospital of Sun Yat-sen University from October 2003 to March 2004. High purification chondrocytes from the knee joints of 3-month-old swine were obtained by enzyme digestion, then the chondrocytes in the third generation were mixed with alginate in a cell density of 5×10^9 L^-1. The suspension was seeded to a 96-wells culture plate with 50 μL at each well and gelled iri calcium chloride solution for 10 minutes, then the cells were further cultured in DMEM medium after adding volume fraction 0.1 fetal bovine serum. Four weeks later, cell appearance, collagen Ⅱ exPression and chondrogenesis ability were evaluated with HE, Masson and immunohistochemical staining. RESULTS： ①Chondrocytes of monolayer culture were polygon, and collagen type Ⅱ was expressed in cytoplasm. Alginate gels and chondrocytes showed perfect gomphosis after mixed. ②HE staining appealed that the chondrocytes of young swine generated and became globular or island in alginate gels. Masson staining showed that collagen among cartilage lacuna was dyed in green color around chondrocytes. Immunohistochemical staining showed that the island of chondrocytes and its surrounding matrix was red, and collagen Ⅱ was excreted massively by chondrocytes. CONCLUSION： Young swine-derived chondrocytes generate and proliferate well when cultured in alginate, so their compound could be used to construct tissue-engineered cartilage in vitro.