多尺度仿骨结构壳聚糖/磷灰石复合材料的按需构建及其细胞共培养

In order to accelerate critical size bone defect reconstruction, on-demand construction and indirect contact cell co-culture in chitosan/apatite composites(CS/HA) with multiscale biomimetic bone structure were developed inspired by the multiscale concentric layer structure and multiple cells of nature bone. Programmable concentration wave and UV photolithography are adopted to achieve the on-demand concentric layer patterns to mimic the lamellar structure of bone and the protein/cell laden chitosan microgels with concentric pattern to mimic the osteon structure. The quantitative regularities of the shape, alternate periodicity, amplification and alternate soaking sequence to modulate the programmable patterns and multiscale orientated porous structures of CS/HA would be established. The mechanism understanding of concentric pattern formation during non-equilibrium gelation process would be demonstrated through the spatial and temporal distribution of hydroxide ions simulated by reaction diffusion model. The on-demand shape, size and pattern array of dentin matrix protein 1(DMP1)/cell laden chitosan microgel with biomimetic osteon structure would be fabricated by UV photolithography with excellent cell viability in order to provide the concentric pattern structure and hydrogel based microenvironment for cells proliferation and differentiation. We would focus on the critical size effect of microgel, function and enhancement effect of biomineral nanoparticles (such as apatite or magnetite nanoparticles) and osteonic differentiation induced by DMP1. Assembling the DMP1/bone mesenchymal stem cells (BMSC) laden microgel into the hollow structure of the tailor-made lamella like CS/HA seeding with human umbilical vein endothelial cells (HUVEC), we would try to address the possible effect of multiscale structure,DMP1 and HUVEC on the induced osteoblast differentiation from BMSC and illustrate the cell/cell interaction of different cells and cell/hydrogel. The multiscale structure chitosan/apatite and indirect 3D cell co-culture would boost the development of vascularized bone tissue engineering and provide the fundamental understanding of critical size bone defect reconstruction.

为实现大尺寸骨缺损的快速修复，基于骨组织多尺度同心层状结构和细胞多样性的特点，采用程序化浓度波和光刻技术分别构建可按需调控的仿环骨板结构CS/HA和原位负载蛋白质/细胞的仿骨单位结构壳聚糖微凝胶。研究浓度波的形状、周期性和振幅对仿环骨板结构参数和取向孔道的调控规律，建立仿环骨板结构参数与力学性能之间的定量关系。采用光刻技术构建形状可控、尺寸可调且原位负载细胞/牙本质基质蛋白1（DMP1）的微凝胶，利用磁场诱导组装获得三维仿骨单位结构组装体。研究微凝胶的尺寸效应和DMP1促进细胞粘附与诱导成骨分化作用。将负载血管内皮细胞的CS/HA与原位负载DMP1/骨髓间充质干细胞的微凝胶组合建立三维细胞共培养平台，研究多尺度仿骨结构、DMP1和血管内皮细胞对干细胞定向诱导分化的影响和血管化作用，阐明DMP1刺激下细胞/水凝胶的交互作用，这将为血管化骨组织工程和诱导大尺寸骨缺损修复提供新途径与实验依据。

为了实现壳聚糖/磷灰石修复临界尺寸骨缺损，基于天然骨组织多尺度同心层状结构，采用程序化浓度波构建可按需调控的仿环骨板结构壳聚糖与壳聚糖/羟基磷灰石复合材料。建立了浓度波的形状、周期性和振幅对仿环骨板结构参数定量调控规律。层数=118/反应周期，厚度=54.43*反应周期-10.42。. 针对壳聚糖存在中性pH下无法水溶、无法UV交联等缺点，采用一步N-酰化合成UV交联壳聚糖—甲基丙烯酰基壳聚糖（N-MAC）。研究发现随着甲基丙烯酸酐与壳聚糖氨基比例的增加，甲基丙烯酰基取代度也会增加。UV交联壳聚糖可通过快速UV辐照（30 s）实现固化，并且UV交联壳聚糖水溶液及其水凝胶都具有较好的细胞相容性。通过UV光刻方法制备出多种图案化微凝胶（包括规则图案以及复杂徽标）。此外UV交联壳聚糖水溶液还可通过皮下注射的方式实现快速皮下UV交联，其水凝胶在体内降解不会引起慢性炎症反应，具有较好的体内生物相容性。负载羟基磷灰石的UV交联壳聚糖复合水凝胶可加速修复临界尺寸的颅骨缺损。. 针对UV交联壳聚糖缺少温度响应性，依次采用自由基接枝与N-酰化两步反应合成温敏UV交联壳聚糖—异丙基丙烯酰胺接枝甲基丙烯酰基壳聚糖。温敏UV交联壳聚糖水凝胶具有温度引发的可逆体积收缩与膨胀性能。研究表明可通过温敏单元NIPAM的接枝量来调控相转变温度以及水凝胶的溶胀性能。将具有光热效应的碳纳米片负载于温敏UV交联壳聚糖水凝胶中，可实现近红外光(NIR)引发的水凝胶体积收缩。将模型药物多柔比星(DOX)负载其中，可实现NIR引发的DOX按需释放行为。. 针对碳纳米点荧光探针存在合成产率低（小于50%）的缺点，分别将含有碳碳双键的壳聚糖与含有碳碳双键小分子/壳聚糖前驱体通过水热碳化法合成碳纳米点。研究发现碳碳双键的含量与合成产率呈现正相关性，最高产率可达85.9%。合成出的碳纳米点由于氮元素的掺杂使得其具有较宽的发射波长（380~550 nm）。碳纳米点荧光探针容易在斑马鱼的肝脏、卵黄以及消化系统富集，并可通过消化系统代谢出去，具有较好的体内生物相容性。此外研究发现碳纳米点还可通过下调内源与外源ROS，在体内起到氧化应激的保护作用。为了赋予监测壳聚糖体内降解，以碳纳米点作为荧光探针，可赋予UV交联壳聚糖水凝胶以红色荧光发射。作为新型荧光探针，碳纳米点具有低光致漂白性能以及良好的生物相容性。

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