小肠黏膜下层组织膜与骨髓基质干细胞联合培养的实验研究

Study on the osteogenesis ability of co-culturing bone marrow stromal cells (BMSCs) and small intestinal submucosa

目的:检测骨髓基质干细胞(BMSCs)与小肠黏膜下层(SIS)复合培养的生物相容性及成骨能力。方法:山羊BMSCs体外成骨诱导至第3代,接种到预湿的SIS上复合培养,以单纯BMSCs相同条件下培养作为对照。通过相差显微镜、扫描电镜及改进的HE染色等方法观察细胞在材料中的生长情况。MTT法检测BMSCs在材料上的增殖活性,碱性磷酸酶(ALP)活性测定及裸鼠皮下异位成骨实验评价其成骨能力,采用配对资料t检验进行统计学处理。结果:扫描电镜及组织学观察发现,细胞材料复合后,BMSCs在SIS上能够良好地粘附和增殖;定量法检测碱性磷酸酶活性显示,细胞加材料组OD值为0.683±0.044,单纯细胞组OD值为0.696±0.039,ALP及MTT测定结果均证实,BMSCs细胞活性及成骨性能不受SIS的影响,与对照组相比,差异无统计学意义(P>0.05)。体内异位成骨实验表明,BMSCs/SIS复合物植入体内后成骨良好,以软骨内成骨方式为主。结论:SIS对BMSCs的生长和成骨功能无影响,具有良好的细胞相容性,且作为一种脱细胞基质的天然膜性材料,SIS具有构建组织工程化骨膜的可行性。

PURPOSE：Using co-culturing of bone marrow stromal cells （BMSCs） and small intestinal submucosa （SIS）, to evaluate the cellular compatibility and the osteogenesis ability as well as the feasibility of SIS to serve as a scaffold in tissue engineering. METHODS： Bone marrow was aspirated from the healthy adult goat and culture-expanded in vitro by the method of total marrow. The third passage cells were transplanted into SIS after being induced to differentiate into osteoblasts for 4 days and then seeded into SIS for 3 to 10 days. BMSCs alone were cultured at the same condition to act as controls. The cellular morphology and function（attachment,proliferation and differentiation） and the ectopia osteogenesis in vivo were assessed separately by means of phase contrast microscope,SEM and MrlT,ALP activity, histology detection. The difference between the two groups were analyzed by Student＇s t test statistically. RESULTS： The BMSCs could adhere to SIS and proliferate and grow on the surface of SIS normally. The cellular activity and function were not affected by SIS, no statistical difference was found between the two groups （P〉0.05）. The BMSCs/SIS compounds had good abilities of ectopia osteogenesis in vivo, and with time going on new bone formation increased. There was no bone formation in the control group. The endochondral bone formatin was the main pattern of osteogenesis. CONCLUSIONS： As a natural, absorbable, acellular scaffold, SIS is biocompatible and can be used as a tissue engineering scaffold. And for a membrane feature, there is the possibility to construct tissue engineered bone membrane with SIS. Supported by Shanghai Leading Academic Discipline Project （Grant No.Y0203）.