人脂肪间充质干细胞与生物材料共混物三维打印体的体内成骨

Osteogenesis of human adipose-derived mesenchymal stem cells-biomaterial mixture in vivo after 3D bio-printing

目的:构建人脂肪间充质干细胞(human adipose-derived mesenchymal stem cells,h ASCs)-生物材料共混物三维生物打印体,检测其体内成骨能力,初步建立将细胞共混物三维生物打印技术应用于体内成骨的技术路线。方法:以P4代h ASCs作为种子细胞,进行成骨向诱导,并采用碱性磷酸酶(alkaline phosphatase,ALP)染色和茜素红矿化结节染色检测其成骨向分化的能力。将种子细胞加入20 g/L海藻酸钠和80 g/L明胶混合物(细胞密度约为1×106个/m L),采用Bioplotter三维生物打印机(德国Envision公司)进行打印,获得细胞-海藻酸钠-明胶共混物打印体,采用活-死细胞双荧光染色法观察打印体中细胞存活率,随后对打印体进行1周成骨诱导培养,作为实验组;另打印不含细胞、只含海藻酸钠-明胶溶胶的三维打印体作为对照组。将实验组和对照组打印体植入裸鼠背部皮下,于植入后6周取出样本,采用苏木精-伊红(hematoxylin-eosin,HE)染色、马松(Masson)三色法染色、免疫组织化学染色和Inveon微型CT(Micro CT)检测打印体样本的成骨情况。结果:打印体中细胞存活率达89%±2%,打印体植入6周后取出,对照组植入打印体大部分发生降解,形态不规则,为无定型凝胶状,而实验组打印体基本保持原有大小,质地坚韧。HE染色和马松三色法染色结果显示,植入6周后,实验组打印体中有类骨组织形成,并有血管长入;免疫组织化学染色结果显示,骨钙素抗体表达成阳性;Micro CT结果显示,实验组密度较高,新生骨容积为18%±1%。结论:h ASCs共混物三维生物打印体可在裸鼠体内异位成骨,细胞共混物三维生物打印技术应用于体内成骨的技术路线是可行的。

Objective：To construct human adipose-derived mesenchymal stem cells （hASCs）-biomate- rial mixture 3D bio-printing body and detect its osteogenesis in vivo, and to establish a guideline of osteogenesis in vivo by use of 3D bio-printing technology preliminarily. Methods：P4 hASCs were used as seed cells, whose osteogenic potential in vitro was tested by alkaline phosphatase （ALP） staining and alizarin red staining after 14 d of osteogenic induction. The cells were added into 20 g/L sodium alginate and 80 g/L gelatin mixture （cell density was 1 × 10^6/mL）, and the cell-sodium alginate-gelatin mixture was printed by Bioplotter 3D bio-printer （Envision company, Germany）, in which the cells＇ survival rate was detected by live-dead cell double fluorescence staining. Next, the printing body was osteogeni- cally induced for 1 week to gain the experimental group; and the sodium alginate-gelatin mixture without cells was also printed to gain the control group. Both the experimental group and the control group were implanted into the back of the nude mice. After 6 weeks of implantation, the samples were collected, HE staining, Masson staining, immunohistochemical staining and Inveon Micro CT test were preformed to analyze their osteogenic capability. Results： The cells＇ survival rate was 89% ± 2% after printing. Six weeks after implantation, the samples of tile control group were mostly degraded, whose shape was irregu- lar and gel-like; the samples of the experimental group kept their original size and their texture was tough. HE staining and Masson staining showed that the bone-like tissue and vessel in-growth could beobserved in the experimental group 6 weeks after implantation, immunohistochemical staining showed that the result of osteocalcin was positive, and Micro CT results showed that samples of the experimental group had a higher density and the new bone volume was 18% ±1%. Condusion：hASCs-biomaterial mixture 3D bio-printing body has capability of eetopic bone formation in nude mice, and it is feasible to apply cells-biomaterial mixture 3 D bio-printing technology in the area of bone formation in vivo.