摘要 目的 制备外观圆整、分散性好、粒径均匀的明胶微球，构建EDC交联明胶微球支架，探索该支架的成骨细胞相容性，为构建组织工程化骨奠定基础。 方法 应用油包水单乳液法制备明胶微球，筛选合适粒径微球。并用EDC/NHS法交联处理微球备用，将小鼠胚胎成骨细胞前体细胞MC3T3-E1与交联处理的明胶微球共培养作为EDC交联明胶微球组；MC3T3-E1培养于细胞培养板作为标准对照组。于接种培养1 d后采用荧光标记的鬼笔环肽进行F-actin染色，培养1、2、3 d后，分别采用活/死细胞荧光染色法及MTT法检测细胞增殖情况，探索EDC交联明胶微球支架上成骨细胞黏附、伸展情况及细胞增殖活力。 结果 制得的明胶微球，外观圆整，分散性好，粒径分布集中。MC3T3-E1可以黏附于明胶微球支架，伸展良好，增殖活跃，表现出良好的细胞活力。 结论 EDC交联明胶微球支架具有良好的细胞相容性，有望成为一种新型的骨组织工程支架以及颅骨缺损修复材料。 Abstract： Objective To prepare gelatin microspheres with a smooth surface, good dispersity, and a consistent particle size, to construct covalently cross-linked gelatin microsphere scaffold using EDC and investigate its compatibility with osteoblasts, and to lay a foundation for constructing tissue-engineered bone. Methods The water-in-oil single emulsion method was used to prepare gelatin microspheres, and the microspheres with a proper particle size were screened out. The selected microspheres were cross-linked by EDC/NHS. Mouse embryo osteoblast precursor cells (MC3T3-E1) co-cultured with cross-linked gelatin microspheres were selected as covalently cross-linked gelatin microsphere scaffold group, and MC3T3-E1 cells cultured in a cell culture plate were selected as standard control group. Fluorescein-labeled phalloidine was used for F-actin staining at 1 day after seeding, and after 1,2, and 3 days of culture, live/dead fluorescent staining assay and MTT assay were performed to evaluate cell proliferation, as well as osteoblast adhesion, stretching, and cell proliferation on the covalently cross-linked gelatin microsphere scaffold. Results The gelatin microspheres prepared by the water-in-oil single emulsion method had a smooth surface, good dispersity, and a consistent particle size. MC3T3-E1 cells showed good adhesion to the gelatin microsphere scaffold, with good stretching and viability. Conclusion The covalently cross-linked gelatin microsphere scaffold prepared by EDC has good osteoblast compatibility and is expected to become a new type of material for bone tissue engineering scaffold and cranial defect repair
