电纺I型胶原/钛酸钡复合仿生骨膜制备及其压电效应通过L型钙通道促进BMSCs成骨分化的机制研究

Periosteum plays an important role during bone regeneration. Construction of highly osteogenic periosteum is the bottleneck of its application in bone defect repair, thereby being a research hotspot. Osteogenic property of the current artificial periosteum relies mainly on the combination with growth factors, whereas the osteogenic effect of physical factors such as electrical stimulation is less concerned. We have previously prepared the piezoelectric PU/PVDF scaffolds by electrospinning, and confirmed its piezoelectric effect on the functions of NIH3T3 cells. Due to the promotive effect of electrical stimulation on bone regeneration, we speculate that scaffolds with piezoelectricity can be used as artificial periosteum, and induce osteogenesis through the piezoelectric effect. In this study, we plan to improve the composition of the existing piezoelectric scaffolds, and prepare the electrospun Colla1/BaTiO3 hybrid bionic periosteum. We will investigate the biocompatibility of the bionic periosteum and its promotive effect on osteogenic differentiation of BMSCs by cell and molecular biology experiments. We will then assess the ectopic osteogenic ability of the bionic periosteum in the subcutaneous tissue of nude mice, and evaluate the effect of the bionic periosteum on bone defect repair in a rabbit radius defect model by histological, imageological and biomechanical approaches. Further, we will explore the probable mechanism of the piezoelectric effect promoting osteogenic differentiation of BMSCs through L-tpye calcium channel. This study provides a new strategy for the treatment of bone defect by artificial bone grafting.

骨膜在骨再生中的作用不可或缺，高成骨诱导性骨膜的构建是其在骨缺损修复应用的瓶颈和研究热点。现有人工骨膜的诱导成骨主要依赖于生长因子的复合，而对电刺激等物理因素的成骨作用则关注较少。我们前期通过静电纺丝制备了具有压电性的PU/PVDF细胞支架，证实了其压电效应对NIH3T3细胞功能的影响。由于电刺激能够促进骨再生，我们推测将具有压电性的细胞支架用作人工骨膜，可望利用压电效应发挥诱导成骨的作用。本课题拟改进原有压电细胞支架的成分，制备电纺I型胶原/钛酸钡复合仿生骨膜，体外通过细胞及分子生物学实验考察该仿生骨膜的生物相容性及其压电效应促进BMSCs成骨分化的作用，并利用组织学、影像学及生物力学等方法，分别在裸鼠皮下和兔桡骨缺损模型评价仿生骨膜的异位成骨能力及在体修复骨缺损的效果，进而探讨压电效应通过L型钙通道促进成骨分化的可能机制。本课题可为人工骨移植治疗骨缺损提供一种新策略。

骨膜在骨再生中起重要作用，构建具有高成骨诱导性的骨膜是骨缺损修复的研究热点。现有人工骨膜诱导成骨主要依赖于生长因子的复合，而对电刺激等物理因素的成骨作用则关注较少。压电材料可在外力作用下产生电效应。由于电刺激能够促进骨再生，推测将压电材料用于制备人工骨膜，可望利用压电效应发挥诱导成骨的作用。本课题计划通过静电纺丝制备一种I 型胶原/钛酸钡复合仿生骨膜并进行相关表征，进而考察其压电效应促进 BMSCs 成骨分化的作用及可能的分子机制，最后评价仿生骨膜在体修复骨缺损的效果。.目前已经制备了电纺I型胶原/钛酸钡、PLLA/钛酸钡等可用作仿生骨膜的备用支架材料，由于I型胶原电纺支架的抗水溶性较差，因此调整研究计划，在后续实验中使用电纺PLLA/钛酸钡作为仿生骨膜。对仿生骨膜的微观形貌、压电性、降解性、力学性能、细胞相容性等方面进行了表征。研发了一种低强度脉冲超声发生装置，该装置主要由超声发生器和6孔细胞培养板底座两部分组成，可以通过超声波激发压电材料产生压电效应。通过体外细胞实验，发现由超声激发的压电效应可以使 BMSCs中BMP2、RUNX2、BGLAP、ALPL等成骨标志物的表达增加。.本研究首次将压电材料应用于仿生骨膜的构建，并为有效激发其压电效应研发了一种低强度脉冲超声发生装置。证实了PLLA/钛酸钡仿生骨膜在超声激发下可以促进 BMSCs 的成骨分化。所设计的低强度脉冲超声发生装置为压电生物材料压电性的可调节性激发提供了保障，填补了压电生物材料难以实现可控性压电激发的技术空白。