径向Denosumab / P34HB电纺支架的制备及其在下颌骨骨缺损修复中的基础研究

Find the suitable scaffold material is always a difficult problem in the study of bone tissue engineering. How to design and prepare bone scaffold with biomimetic properties is also a difficult problem for the bone reparation project. The project intends to fabricate radial Denosumab / P34HB electrospun scaffold with the properties of structure mimic, composition mimic and stiffness mimic via the electrospinning technique, at the same time, the activity of antibody was maintained by the preparation of BSA nanoparticles loaded with Denosumab. The radial structure of the scaffold not only could induce the adhesion, proliferation and differentiation of MSCs, but also has a strong effect on the induction of bone formation. More important, the structure could improve the bone contact area of the implant and it could also increase the locking and fixation of the implant and the bone. The way of implanting the scaffold into the bone effect is stacking layer by layer and to construct a three dimensional structure which is similar to the body environment, so as to increase the relationship between the cell and the scaffold, the cell and the cell, and is beneficial to the formation of the new bone. At the same time, the slow release of Denosumab in structure is more beneficial to bone formation. The project propose a comprehensive study in.physical and chemical properties of scaffold, the biocompatibility, induced cells growth and differentiation, in vivo degradation and the rabbit mandibular chest-like bone defect reparation, etc. The project would not only provide the new ideas and methods to design and fabricate the bone repair scaffolds, but also offer the helpful reference for the research of bone tissue engineering.

寻找适宜的支架材料一直是骨组织工程研究的难题。如何设计和制备出有仿生性质的骨三维支架是目前骨修复工程的难题。本项目拟采用静电纺丝技术构建新型的具有结构、成分及力学仿生的径向Denosumab / P34HB电纺支架，采用制备载Denosumab的BSA纳米颗粒的方式保持抗体的活性。支架表面径向的结构可以诱导MSCs的黏附、增殖和分化，并对骨形成具有很强的诱导作用，不但能提高植入体的骨接触面积，而且可以增加植入体与骨的锁合及固位。支架植入缺损区的方式为层层叠加，构建成与机体环境相似的三维结构，从而增加细胞与支架，细胞与细胞之间的联系，更有利于新骨的形成。同时，支架中Denosumab的缓慢释放更有利于骨的形成。本项目拟全面研究该支架的理化性质、生物相容性、细胞诱导、体内降解及兔下颌骨箱装骨缺损修复等。本项目将为骨缺损修复支架的设计和构建提供新的思路和方法，为骨组织工程的研究提供有益的参考。

随着生物材料和加工技术的发展，骨组织工程技术在促进骨缺损修复的治疗方法中得到了广泛关注。寻找适宜的支架材料、构建和制备出具有仿细胞外基质结构的三维骨组织工程支架仍然是目前骨组织工程的难点。本研究的目的是运用静电纺丝技术和浸泡法制备出负载有卵磷脂的亲水性径向P34HB电纺支架，表征其各项理化性能，探究其生物相容性及成骨性能，为骨组织工程支架的设计和构建提供新的思路和方法。. 本课题采用静电纺丝和浸泡法分别构建了无序的P34HB电纺支架（PR组）、径向P34HB电纺支架（PA组）、亲水性无序的P34HB电纺支架（PR/L组）和亲水性径向P34HB电纺支架（PA/L组），并对各类支架的理化性能进行表征。提取、培养和鉴定BMSCs，将细胞接种于各类支架上，检测细胞增殖、黏附及形貌，采用划痕实验观察卵磷脂对细胞迁移的影响。并同时检测接种于不同支架上的BMSCs的ALP活性，细胞外基质矿化情况，及成骨相关基因的表达水平。对黏蛋白Vinculin进行染色，观察早期黏附时黏着斑情况及黏附相关基因的表达水平。动物实验部分采用大鼠背部皮下植入模型和大鼠颅骨缺损模型，观察支架体内相容性、血液分析、体内重要脏器分析及新骨形成情况。结果表明卵磷脂显著改善了支架的疏水性能，PA/L组具有良好的体外生物相容性，BMSCs在PA/L支架上的增殖更好，卵磷脂可以提高细胞迁移速率，支架上的部分细胞长入纤维支架内部，形态呈现细长且高度分支的“类成骨细胞”样，细胞表现了较高的ALP活性、细胞外基质矿化和成骨基因表达。此外，PA/L可以更好的调控细胞黏附，上调黏附相关的基因表达。PA/L支架在大鼠背部皮下表现了良好的体内生物相容性。骨缺损处的PA/L植入未引起重要脏器及血液的显著性功能变化，并可以促进骨缺损处的新骨形成。. 采用静电纺丝技术制备得到径向排列P34HB支架，径向排列纤维形貌和支架上负载的卵磷脂发挥协同促成骨作用，在体外能更好的促进BMSCs成骨向分化，同时在体内也能促进颅骨的骨缺损修复。因此，亲水性径向P34HB电纺支架在骨组织工程方面具有良好的应用前景。