兔血管内皮细胞和诱导成骨细胞在可注射纳米材料上的共培养

Co-culture of rabbit vascular endothelial cells and rabbit bone marrow stem cells induced osteoblasts on injectable nano-materials

目的:将可注射纳米骨材料与共培养的兔肾血管内皮细胞和兔骨髓间充质干细胞诱导的成骨细胞复合,并构建可注射细胞型纳米组织工程骨,观察它们体外培养的相容性。方法:实验于2003-09/2004-11在苏州大学附属儿童医院完成。①实验材料:取16周龄雄性新西兰大耳白兔,体质量1.5kg左右。②实验方法:麻醉后抽取兔骨髓,用淋巴细胞分离液分离出其中的间充质干细胞,在含体积分数为0.15牛血清的RPMI1640液中培养。骨髓间充质干细胞在条件培养基中,7d后可见细胞变为多边形,碱性磷酸酶和Ⅰ型胶原染色阳性,形成钙结节,表现成骨细胞分化特点。采用三步梯度筛网法,获得肾血管球,5g/L胶原酶Ⅳ37℃消化15～20min,离心沉淀获取血管内皮细胞,在含体积分数为0.15小牛血清的M199中培养。③实验评估:免疫组织化学法进行第Ⅷ因子相关抗原鉴定,透射电镜观察细胞浆Weibel-Palade小体,间接免疫荧光法检测CD31、CD34及CD44的表达。将共培养的兔肾血管内皮细胞、成骨细胞与可注射纳米骨材料体外复合培养,进行形态学观察和功能测定。结果:①在一定培养条件下成功诱导兔骨髓间充质干细胞向成骨细胞分化。②培养的血管内皮细胞免疫组织化学法检测第Ⅷ因子相关抗原阳性,透射电镜观察到细胞浆中的Weibel-Palade小体,间接免疫荧光法检测CD31、CD34表达阳性,CD44阴性。③共培养的兔肾血管内皮细胞、成骨细胞在可注射纳米骨材料上生长、增殖良好,细胞活性和碱性磷酸酶活性未受到影响。结论:可注射纳米骨材料具有良好的细胞相容性,可作为骨组织工程理想的可注射载体材料。

AIM：To explore the cellular compatibility of the injectable nano-hydroxyapatite/collagen （nHAC） with rabbit renal vascular endothelial cells （RVECs） and osteoblasts （OB） induced from rabbits bone marrow stem cells in vitro, and construct the injectable cytotype tissue engineered bone. METHODS： The experiments were carried out in Soochow University Children＇s Hospital from September 2003 to November 2004. ①The 16-week old New Zealand rabbit weighed 1.5 kg were adopted in this study. ②Rabbit bone marrow were obtained from rabbits by aspiration under anesthesia, and bone marrow mesenchymal stem cells （MSCs） were separated by percoll gradient centrifugation and incubated in RPMI1640 containing 15% fetal bovine serum （FBS）. After 7-day induction in conditional culture, MSCs became polyedged morphologically, and the cells revealed the characteristics of osteogenic differentiation with strong staining for alkaline phosphatase activity, immunochemical expression of type Ⅰ collagens, and formation of mineralized MSCs nodules. Rabbit RVECs were incubated in M199 containing 15% FBS and harvested from renal glomerruli by three steps grade filtrate net, followed by digestion with 5 g/L collagenase at 37 ℃ for 15- 20 minutes. ③RVECs were identified by immunohistochemical method and Weibel-Palade bodies were observed under transmission electron microscope. The antigens CD31, CD34, .and CD44 were detected by indirect immunofluorescence methods. The RVECs and OB were co-cultured in vitroon the injectable nHAC, their morphology and biology were studied. RESULTS： ①Rabbit bone marrow MSCs were induced to differentiate into OB under certain culture.②Factor Ⅷ related antigen was positive for RVECs by immunohistochemical method, and Weibel-Palade bodies were identified under transmission electron microscope. CD31 and CD34 were positive, while CD44 was negative for indirect immunofluorescence methods. ③The co-cultured RVECs and OB grew and proliferated normally with the injectable nHAC. The cellular viability and alkaline phosphatase activity were not influenced by nHAC. CONCLUSION： The injectable nHAC has a good biocompatibility and may be used as ideal biomaterial in bone tissue engineering.