生物打印技术构建血管神经化组织工程骨的研究

血供不足和缺乏神经支配是组织工程骨移植失败的重要原因，在组织工程骨中同步建立血管化和神经支配至关重要。本项目拟通过生物打印技术同步构建带有血管化和神经支配的组织工程骨，建立基于血管和神经同步构建的生物打印技术。通过制备具有骨组织适配性的生物墨水，并结合微流控技术制备的细胞微囊，实现打印过程中的细胞活性维持。利用同轴生物打印技术，以含有干细胞微囊、内皮细胞和神经细胞的生物墨水为外层，载内皮细胞的明胶墨水为内层，打印具有牺牲内层的皮/芯结构支架。通过调控外层结构中的不同细胞功能，模拟骨生长的血管化和神经支配的微环境，以生成毛细血管和神经交织生长的骨组织。内层含内皮细胞的明胶墨水在打印时提供支撑，培养时明胶溶解并释放内皮细胞，最终形成较大尺寸的血管通道。本项目将对打印的组织工程骨进行体外培养和评价，并通过动物实验验证其在体内的血管化、神经化和骨修复潜能，探明血管化、神经化对其成骨的影响和机理。

Insufficient blood supply and lack of innervation are important reasons for the failure of tissue engineered bone implants, and thus simultaneous construction of vascularization and innervation in tissue engineered bone implants is of crucial importance. This study aims to construct tissue engineered bone implants with vascularization and innervation simultaneously using 3D bioprinting technology, and build multi-cell synchronous printing technology for engineering of the vascularized and innervated bone implants. To this end, the cell encapsulation for osteogenic stem cells was prepared using microfluidic technology, which can facilitate to achieve high cell viability during bioprinting. The coaxial 3D bioprinting was used to print the cell-laden sheath/core tissue structure with sacrificial inner layer. During the bioprinting, the bioink carrying stem cell encapsulation, endothelial cells and nerve cells was used as the outer layer, which can recapitulate the microenvironment in native skeletal tissue by regulating the functions of different cells, and thus constructing a vascularized and innervated tissue engineered bone. While the gelatin bioink loading with endothelial cells served as the sacrificial inner layer, which provides structural support for printing and can be dissolved to generate channels, and the released endothelial cells can deposit and adhere to the channels automatically, eventually forming vascular channels in the engineered tissue. The printed tissue engineered bone will be cultured in vitro and evaluated by animal tests, and the effect of vascularization and innervation on the osteogenic properties and underlying mechanisms will be investigated.