骨髓基质干细胞与PDPB体外构建组织工程骨的适宜条件

Optimal condition of in vitro constructing tissue-engineered bone with bone marrow stromal cells and partially deproteinised bone

背景：骨组织工程支架材料中PDPB是一种较为理想的骨移植材料，目前多采用体外培养的骨髓基质干细胞与支架材料构建组织工程骨后修复骨缺损。目的：实验拟验证骨髓基质干细胞与PDPB体外构建组织工程骨的最佳浓度及移植入体内的最佳时机。设计．时间及地点：随机分组设计，对照观察，于2007—10／12在中国科学院动物研究所完成。材料：2周龄日本大耳兔，体质量2．0kg左右，用于培养骨髓基质干细胞。取新鲜的猪椎骨制备PDPB。方法：传代培养骨髓基质干细胞，矿化液对兔骨髓基质干细胞进行分化鉴定后，取不同浓度（1×10^10L^-1、5×10^9L^-1、1×10^9L^-1、5×10^8L^-1）的兔骨髓基质干细胞与PDPB复合体外构建组织工程骨。主要观察指标：倒置显微镜下观察细胞的生长形态：扫描电镜观察骨髓基质干细胞在PDPB上的黏附情况：四甲基偶氮唑盐法检测各浓度骨髓基质干细胞在PDPB上的生长情况。结果：①原代培养24h部分细胞开始贴壁，6d后贴壁生长的细胞数量增多，10--12d集落逐渐增大，并融合为单层。经矿化液诱导培养21d后，行矿化沉积茜素红染色及碱性磷酸酶细胞化学染色，可见大量细胞呈碱性磷酸酶阳性及大量钙结节形成。②扫描电镜观察骨髓基质干细胞在PDPB上黏附良好，第8天时细胞和材料结合紧密。第10天细胞呈梭形生长，部分细胞突起翘起。③各浓度组骨髓基质干细胞在PDPB上的生长曲线形态基本相同，呈“S”形。2-6d细胞开始在支架材料上大量增殖，第6天后细胞活力逐渐降低，其中5×10^9L^-1浓度骨髓基质干细胞复合PDPB后细胞可以很好的黏附，细胞活性好。结论：骨髓基质干细胞和PDPB复合的最佳浓度是5×10^9L^-1，移植的最佳时机为6d。

BACKGROUND： The partially deproteinised bone is one of ideal scaffold materials of bone transplantation. Presently, in vitro cultured bone marrow stromal stem ceils and scaffold material are used to construct tissue-engineered bone for bone defect. OBJECTIVE： To explore the optimal cell seeding density and the optimal occasion to transplant the tissue-engineered bone with bone marrow stromal cells and scaffold material. DESIGN, TIME AND SETTING ： The randomized grouping control experiment was performed at the Institute of Zoology, Chinese Academy of Sciences from October to December in 2007. MATERIALS： Japanese big ears rabbits, two weeks old, weighing about 2.0 kg were used to culture bone marrow stromal stem cells Fresh pig vertebrae were used to make partially deproteinised bone. METHODS： Bone marrow stromal stem cells were cultured and identified by the mineralized liquid. Rabbit bone marrow stromal stem cells at different densities （1 × 10^10 L^-1, 5× 10^9 L^-1, 1 × 10^9 L^-1, 5× 10^8 L^-1） were seeded onto partially deproteinised bone to construct tissue-engineered bone in vitro. MAIN OUTCOME MEASURES： Morphology was observed under an inverted microscope. The adhered information of bone marrow stromal stem cells on partially deproteinised bone was observed with a scanning electron microscope. The adhesive and proliferation conditions of bone marrow stromal stem cells were evaluated by MTT. RESULTS： Some cells began to adhere after 24 hours of primary culture, and more cells began to adhere 6 days later, and the colony became larger 11- 12 days later and was confluent. After 21 days of mineralized liquid induction, cells received the alizarin bordeaux staining and the alkaline phosphatase staining, showing alkaline phosphatase-positive cells and a large population of calcium noduses. Bone marrow stromal stem cells could adhere to partially deproteinised bones commendably. At day 8, cells were closely connected to materials. At day 10, ceils were spindle, and some with processes. Growth curves of bone marrow stromal stem cells on the partially deproteinised bone were similar at different densities, presenting ＂S＂ form. Bone marrow stromal stem ceils proliferated from day 2 to day 6 on the partially deproteinised bone. Cell activity decreased at day 6. Bone marrow stromal stem cells at a density of 5 × 10^9 L^-1 had a good adherence and activity on the partially deproteinised bone. CONCLUSION： The optimal seeding density of constructing tissue-engineered bone with bone marrow stromal cells in vitro is 5 × 10^9 L^-1, and the optimal occasion to transplant is at day 6.