基于定向光控细胞片技术的血管化组织工程骨膜的构建及相关机制研究

The periosteum provides vital blood supply to the cortical bone and acts as the source of bone-forming cells. It plays a critical role in bone homeostasis and regeneration. However, the natural periosteum can’t be widely used in clinical practice because of the limited availability, and previous studies on tissue-engineered periosteum constructs have failed to accurately reproduce the structure and function of the natural periosteum. Our previous studies confirmed that light-induced anisotropic fibroblasts sheets could secrete aligned extracellular matrix and had better pro-angiogenic effects; anisotropic mesenchymal stem cell sheets retained multidirectional differentiation capacity with enhanced osteogenic potential. On this basis, the aim of this study is to explore the feasibility of constructing a double-layer vascularized tissue-engineered periosteum with aligned extracellular matrix by light-induced anisotropic cell sheet technology and photosensitive gel system. The osteogenesis and angiogenesis effects of biomimetic periosteum combined with 3-D printed PCL scaffold will be evaluated in vitro and in vivo. Meanwhile, the role of anisotropic cell sheets and aligned extracellular matrix in the formation of 3-D microvascular network of biomimetic periosteum was studied. The role of YAP/TAZ-based mechanotransduction pathway in cell alignment and differentiation was deeply studied. The molecular mechanism of aligned extracellular matrix promoting ECs vascularization was elaborated. We believe these work will elicit a deeper understanding of the structure and function of the natural periosteum, and provide experimental basis and theoretical support for the clinical application of the new tissue engineered periosteum.

骨膜既是骨皮质的血供来源，也是骨生发的细胞来源，在骨稳态和再生中起着关键作用。但天然骨膜来源有限无法广泛应用于临床，而现有的骨膜组织工程研究尚不完善。申请人前期研究证实，定向成纤维细胞片可分泌有序的细胞外基质并能促进ECs血管化；定向MSCs细胞片保留多向分化潜能且成骨能力得到强化。在此基础上，本研究拟基于定向光控细胞片培养技术探讨构建具有仿生细胞外基质的双层血管化组织工程骨膜的可行性，与3D打印PCL支架复合后在体内外评价其成骨及成血管效果。同时，重点研究定向细胞及方向性细胞外基质在仿生骨膜三维血管网形成中的作用，探讨转录调节子YAP/TAZ如何整合机械力信号与可溶性分子以控制ECs行为，阐明定向成纤维细胞片促进ECs血管化的分子机制。本研究有助于加深对天然骨膜的结构和功能的认识，深化材料拓扑结构调控细胞行为的分子机制，为新型组织工程骨膜的临床应用提供实验基础和理论支持。

骨膜在骨缺损的修复中起到重要的作用，但由于天然骨膜来源有限性使其临床应用受限。因此构建仿生骨膜是组织工程骨修复的新方向。天然骨膜内层具有固有定向的拓扑形貌，我们的研究发现二氧化钛纳米点/棒进行光功能化能够实现对细胞的定向排列，且这种光功能化的作用与紫外线辐照能呈剂量依赖性。定向排列能通过细胞骨架的变化调控YAP/TAZ相关信号通路有效促进人间充质干细胞（hMSCs）体外成骨向分化。通过这种光控细胞片技术我们分离得到了定向排列的细胞片及其细胞外基质，同样观察到分离的定向细胞片ALP活性、茜素红S染色以及成骨基因表达提高，RNA测序提示定向细胞片磷脂酰肌醇3激酶（PI3K）-蛋白激酶B（AKT）通路的显著上调，通过抑制表达证实了PI3K-AKT通路参与其细胞骨架相关的成骨分化。此外，与人脐静脉血管内皮细胞（HUVEC）共培养的定向细胞片能促进血管生成。为模拟天然骨膜双层的结构，我们通过数字化光处理打印技术获得适应缺损区域的水凝胶外层，联合定向细胞片对小鼠颅骨缺损模型进行修复，通过Micro-CT、组织学染色发现定向细胞片相较于随机排列的细胞片具有更好的体内修复骨缺损以及血管化的能力。进一步的，我们基于形状记忆聚合物（SMP）将动态变化的拓扑微结构引入骨膜内侧，证明了其在体外对干细胞成骨命运的调控以及体内成骨能力，实现了仿生骨膜的远程调控响应。综上，我们的研究为骨膜组织工程以及骨缺损的修复提供了一种新思路。