人牙髓细胞三维生物打印的初步研究

通过生物打印方法构建含人牙髓细胞(human dental pulp cells,hDPCs)的组织工程三维结构体。探讨对hDPCs进行三维生物打印的可行性。用常规体外培养hDPCs至第4代。将细胞悬液与海藻酸钠-明胶水溶胶混合,制备hDPCs-海藻酸钠-明胶水溶胶共混物。细胞终密度为2×106/mL。用生物打印机根据预先设计的参数进行生物打印,构建组织工程三维结构体。对结构体进行肉眼观察和倒置相差显微镜观察。用钙黄绿素-AM和碘化丙啶双荧光染料对结构体进行染色,激光共聚焦显微镜观察分析结构体中hDPCs的活性。通过生物打印技术的结果可按照预先设计的参数打印出含有hDPCs的网格状的水凝胶结构体,该结构体的大小约为5 mm×5 mm×1.5 mm。倒置相差显微镜下可见结构体内微丝的直径约300μm,微丝间的孔隙相互通连,孔隙大小约350μm×350μm,hDPCs在结构体中呈球形均匀分布。激光共聚焦显微镜观察证实结构体中大部分细胞存活。初步证明hDPCs可耐受生物打印过程。通过研究说明三维生物打印技术构建出了含人牙髓细胞的组织工程三维结构体,实现了hDPCs的生物打印,为生物打印技术应用于牙组织工程奠定了基础。

Preliminary study on three dimensional bioprinting of human dental pulp cells

To fabricate tissue-engineered three dimensional(3D) structures containing hDPCs by bioprinting and investigate the feasibility of 3D bioprinting of human dental pulp cells(hDPCs),the hDPCs were regularly cultured in vitro to harvest cells at passage 4.Alginate and gelatin were mixed to form hydrosol and hDPCs suspension was added into the hydrosol.The mixture was used to fabricate 3D tissue-engineered structures by 3D bioprinting machine according to certain parameters.The 3D structures were observed with inverted phase contrast microscope.Structures were stained with Calcein-AM and propidium iodide dye and laser scanning confocal microscope was applied to assess cell viability.It is resulted that grid-like hydrogel architectures with hDPCs embedded are fabricated by bioprinting technique.The size of the structure is about 5 mm×5 mm×1.5 mm.The structure is composed of vertically staggered microfilaments which are about 350 μm in diameter.Rectangular micro-channels about 350 μm are created in the structure.Human dental pulp cells with high cell viability are homogeneously embedded in these structures.It is conclused that in this study the 3D bioprinting of hDPCs is implement,which lays the foundation for the application of bioprinting technique in the field of tooth tissue engineering.