Osteogenesis of human adipose-derived mesenchymal stem cells-biomaterial mixture in vivo after 3 D bio-printing / 北京大学学报(医学版)

ABSTRACT

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 1 4 d of osteogenic induction.The cells were added into 20 g/L sodium alginate and 80 g/L gelatin mixture (cell density was 1 ×1 06/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 the 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 be observed 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 1 8%±1%.Conclusion:hASCs-biomaterial mixture 3D bio-printing body has capability of ectopic bone formation in nude mice,and it is feasible to apply cells-biomaterial mixture 3D bio-printing technology in the area of bone formation in vivo.