基于MMP酶识别活性多肽调控凝胶降解和细胞铺展空间的基质功能化与骨的组装构建

This research aims to optimize the microenvironments of cell-laden hydrogels and to build osteo-like structure in bottom-up approach by the directed assembly of cell-laden microhydrogels. Based on the characteristics of anchorage-dependant cells (ADCs) like osteoblasts,which require attachment to a substrate to survival, this research will firstly try to establish a microenvironment to supply space for cell spreading and adhensive sites for cell adhension thus to establish cell survival, and then construct the biomimetic structure of bone tissue by cell-laden microhydrogels assmbly. The focus of this study is to manipulate the hydrogle degradation mediated by matrix metalloproteinase family (MMPs) sensitive peptides and the resulted micro-space for cell spreading. A photo crosslinkable hyaluronic acid (HA) will be used as the main network in the hydrogel, and MMPs sensitive peptides will be composited to form the 2nd network in the hydrogel, and then the composite hydrogel is MMPs sensitive degradable. In this system, the rapid degradation of peptides networks mediated by MMPs secreted by cells themselves will creat cavities for cell spreading and reduce the limitation to cells by the hydrogel networks, and the "rigid" networks by HA will promise the little constraction of the space to support cell spreading. The degradation of the hydrogel networks is tunable by the amounts of peptides composited. At the same time, the chemical environment for cell adhension or differentiation can be obtained by modification of HA by RGD peptides and other growth factor-like peptides. Thus a functional matrix for cell adhension, spreading and differentiation will be established in the hydrogels. Based on this optimazation of hydrogel environment, construction of bone tisue will be performed by mimicking the structure of osteon by assembly of cell-laden microhydrogels. Further assembling the osteon-like units, macro bone tissue constructions will be obtained. With microchannels acting as vascular vessels and osteoblasts area acting as bony structure around channels, this assembly is likely to realize structural biomimetic and finally reach the functional biomimetic of bone tissue.

本项目研究用于单元组装构建仿生骨组织的凝胶功能化及骨组织的组装构建。针对成骨细胞等贴壁型细胞的粘附铺展特点，构建适宜于细胞粘附和三维铺展的粘附位点和空间微环境，再利用单元制造和组装技术构建骨的仿生结构。重点研究金属基质蛋白酶（MMPs）识别的可降解活性多肽调控下的凝胶降解行为及微空间构建，以具有光交联活性的透明质酸为主体凝胶，通过具有MMP识别活性的多肽与透明质酸形成双网络结构，获得细胞酶调控降解的水凝胶，在该体系中，多肽网络被细胞酶快速降解降低对细胞的束缚，透明质酸网络的低收缩性提供空间"支撑"作用，从而为细胞铺展提供空间，网络的降解行为可通过降解肽的引入量调整。同时，通过含细胞粘附活性位点等功能性多肽对凝胶的化学修饰，获得凝胶中细胞的粘附及分化功能。在此基础上，结构仿生制造细胞微凝胶单元，组装获得成骨与成血管的功能组合的骨单位，进一步多级组装，为获得仿骨结构和功能的骨组织奠定基础。

本项目探索了用于单元组装构建仿生骨组织的凝胶功能化及骨组织的组装构建。针对成骨细胞等贴壁型细胞的粘附铺展特点，重点围绕构建适宜于细胞粘附和三维铺展的粘附位点和空间微环境，以及基于单元制造和组装技术构建骨的仿生结构展开了研究工作。获得了一系列基于天然高分子水凝胶的甲基丙烯酸化改性的材料体系，其光交联特性方便凝胶的微单元制造与组装，活性双键的引入利于凝胶分子的改性。设计制备了具有细胞粘附活性的多肽片段，以及具有金属基质蛋白酶（MMPs）降解活性的多肽序列，通过活性多肽对凝胶高分子的接种改性，获得了一系列既具有细胞粘附活性又具有细胞分泌酶降解活性的凝胶材料，显著促进了凝胶中细胞的铺展、粘附、增殖和分化等功能表达；研发的材料体系除可用于本研究的组织微制造技术外，还可广泛用于细胞载体、组织工程构建，为组织工程提供新型功能化载体材料。同时，基于微制造技术和“自下而上”组装对类似骨单位的血管内皮细胞/成骨细胞分层分布的仿生结构，实现了微米尺度范围内骨组织的精细结构仿生，对于多种复杂组织的仿生构建提供广泛基础和借鉴，具有重要科学意义和广泛应用前景。