纳米纤维小径人工血管的微生物制造及其结构调控

Development of succedaneous materials of small-diameter blood vessels is always a hot and difficult point in the biomedicine area. In recent years there have been several technology and patents with regard to production of small-diameter artificial blood vessels by using bacterial cellulose (BC), however, disadvantages including low preparation efficiency, material lamination and compliance problems are found. During recent period, the applicant has demonstrated to utilize a unique double-tube bioreactor and independent patent technology to successfully obtain BC tube series, whose diameter and length can be controlled. The preparation technology shows the following advantages: high efficiency, uniform structure and no delamination of BC tubes obtained. However, the mechanical properties including compliance are far from the requirements of practical applications, and biocompatibility of the BC tubes lacked evaluation. In this project, based on the existing technology and methods, the relationship of "cellulose synthesis - nano fiber network structure - mechanical properties" will be investigated thoroughly by using several key technologies such as microelectrode measurement in situ and fluorescent live cell staining. The investigation will be performed from the influence of micro environment in the bioreactor on bacterial livability and biosynthesis of BC, so that the mechanisms behind the relationship between structure and strength will be elucidated, and thereafter strategies for regulating network density and strength of BC tubes would be established. In order to satisfy the requirement that the mechanical properties of artificial blood vessel match the native vascular mechanical performance, double network structural BC tube composites will be constructed and studied. In addition, key purification technology of BC tubes will be explored, and its biocompatibility will be evaluated to lay the foundation for future in vivo experiments in animals.

小径血管替代材料的开发一直是生物医学领域的热点和难点。近年出现了多种利用细菌纳米纤维素（BC）制备小径人工血管的技术和专利，但是存在制备效率低、材料分层和顺应性不足等缺憾。申请人通过双套管生物反应器和自主专利技术成功获得直径及长度可控的系列BC管，不仅制备效率高，而且材料结构均匀，无分层现象，但是顺应性等力学性能仍未达到实际应用要求，也缺乏生物相容性评价。为此，本项目在已有工作基础上，拟通过利用微电极原位测定技术和荧光活细胞染色等关键技术，从微环境影响细菌活度和BC生物合成的机制上，多角度系统研究"纤维素合成-纤维网络结构-力学性能"之间的关系，阐明BC管结构和强度之间的关联机制，建立调控BC管网络密度和强度的技术方法；研究利用双网络结构改善BC管的顺应性等生物力学性能，以满足BC人工血管与天然血管的性能匹配要求；探索BC管纯化共性关键技术，评价其生物相容性，为今后的动物体内实验奠定基础。

血管移植是当前治疗小径血管疾病的有效手段。但是，自体血管来源有限，远不能满足血管替换需求；而临床成功应用于大口径血管的涤纶和膨体聚四氟乙烯等管材，在用于小口径人工血管时存在栓塞和内膜增生等问题。因此临床上仍旧缺乏小口径人工血管。在众多血管材料研究中，细菌纳米纤维素（bacterial nano-cellulose，BNC）因具有独特的理化性质，在人工血管中具有极高的应用潜力吸引了众多学者的关注。目前BNC管的制备领域存在效率低、材料分层和顺应性不足等缺憾，也缺乏微环境合成过程研究和生物相容性评价。本课题通过利用先进的分析技术，研究了微环境影响细菌活度和BNC生物合成的机制；探索了双网络结构对BNC管的增强和改性，制备了BNC/PVA和BNC/CH等复合管，提高了BNC管的机械力学性能，改善了顺应性，可以满足多种人体组织血管的力学要求；建立了BNC管的后续纯化技术，评价了其体外血液和细胞相容性等生物相容性，通过接枝肝素提高了BNC小径管的抗凝血性能和促内皮化效果，为BNC管的动物体内实验奠定了良好的物质基础。结果显示：反应器和菌株类型、培养基碳源浓度、培育条件对BNC小径管的纤维密度、形态形貌、理化性质、力学性能以及生物相容性等均有不同的影响。譬如，生物反应器的类型决定了供氧方式和水平的差异，从而影响微环境溶氧、细菌分布和小径管形态；而且反应器和菌株类型对获得的BNC管的肝素接枝效果也有不同影响。本研究结果对BNC小径管的优化制备和性能改进均具有非常重要的理论指导意义。通过项目的实施，该课题已获得省部级一等学术奖励2项；已申请中国发明专利12项（已获授权1项），申请实用新型专利3项（已获授权2项）；在国内外高水平学术刊物上共发表期刊论文22篇，其中发表在本领域2区以上期刊SCI论文10篇，北大核心/CSCD中文核心期刊上发表论文8篇；撰写英文专著章节1章；项目组成员共参加国际学术会议5次，参加国内学术会议4次，获邀请报告2次；培养研究生9名，其中已毕业博士生2名，硕士生5名，在读研究生2名。