Notch信号通路在含腺苷矿化胶原三维支架促进骨修复中的作用和机制研究

Large bone defects are difficult in self-healing, which will seriously affect the prognosis and life quality of patients. Three-dimensional (3D) bone repair scaffolds can promote the repair of bone defects. However, problems still exist, such as difficult preparation of bionic structures and unsatisfactory biological activity. In the former studies, we have found that calcium phosphate complex minerals with high biological activity can be prepared by reaction between calcium ions and phosphate group released from adenosine triphosphate, which is catalyzed by basic phosphatase. On this basis, we proposed a new strategy for the preparation of enzymatically catalyzed ATP to construct the adenosine-containing 3D mineralized collagen (Ad/CaP@Col) scaffolds for bone repair. First, the chemical composition, structure, biosafety and bioactivity of the Ad/CaP@Col 3D scaffolds will be demonstrated by systematic characterization of physical and chemical properties, as well as in vitro/vivo biological experiments. Then a rat skull defect model will be established to evaluate the remediation effect of Ad/CaP@Col 3D scaffolds on bone defects. Besides, the role and mechanism of Notch signaling pathway in promoting osteogenic differentiation and bone repair by Ad/CaP@Col 3D scaffolds, will be further explored and clarified. In conclusion, this research will provide both experimental and theoretical basis for preparation and translational application of this new biomimetic 3D mineralized collagen scaffold.

大段骨缺损难以自愈，严重影响患者预后和生活质量。三维骨修复支架可促进骨缺损的修复，但通常存在仿生结构制备困难、生物活性不理想等关键问题。申请人前期研究发现，通过碱性磷酸酶催化三磷酸腺苷(ATP)释放的磷酸根与钙离子反应，可制备具有高生物活性的磷酸钙复合矿物。在此基础上，本项目提出了酶催化ATP构建含腺苷矿化胶原(Ad/CaP@Col)三维骨修复支架的新制备策略。拟进行系统的理化性质表征和体外/体内生物学实验，研究Ad/CaP@Col三维支架的化学组成、结构、生物安全性和促成骨分化生物活性。拟利用大鼠颅骨缺损模型评价Ad/CaP@Col三维支架促进骨缺损修复的性能。拟深入探索和阐明Notch信号通路在Ad/CaP@Col三维支架促进成骨分化和骨修复过程中的作用及机制。本项目的顺利开展将为新型仿生矿化胶原三维支架的制备及转化应用提供实验基础和理论依据。

磷酸钙和胶原蛋白等天然骨组织组分的生物材料具有良好的生物相容性，而且可与骨修复相关细胞发生相互作用，从而调节细胞的粘附、增殖和分化性能。包括磷酸钙和胶原蛋白在内的仿生材料的研究受到国内外相关领域学者关注。自体脱细胞脂肪源基质（DAM）主要成分为胶原蛋白纤维。因此，在胶原三维支架基础上进一步改进为DAM代替胶原，制备了矿化DAM（mineralized DAM，mDAM）三维支架，并进一步引进生物活性分子单宁酸（tannin）增强其抗炎作用，制备单宁酸-mDAM（TA@mDAM）三维支架。本项目成功表征了新制备的TA@mDAM三维支架的理化性质：表面形貌、化学组分、孔隙率、力学性能以及降解性能和三维支架的生物安全性。细胞和动物实验证实了TA@mDAM三维支架诱导BMSCs细胞成骨分化和修复大鼠颅骨缺损的效果。进一步研究拟证明激活Notch细胞信号通路是TA@mDAM三维支架诱导BMSCs成骨分化的关键。同时基于大鼠颅骨缺损模型验证TA@mDAM三维支架激活Notch细胞信号通路促进骨修复。本项目的特点在于运用仿生学原理、生物矿化技术和纳米自组装技术，模拟天然骨组织中的成分及结构特征，制备纳米级别高活性矿化胶原三维骨修复材料。区别于传统液相沉淀的制备方法，本项目提出的碱性磷酸酶和ATP分子介导的生物矿化策略，可以制备得到含腺苷的新型矿化胶原三维支架。与传统单纯混合制备的复合胶原骨修复支架相比，将具有更优良的生物活性和促进骨修复的性能。通过系统的体内和体外实验，本项目证明了TA@mDAM三维支架的理化性质、生物学效应，特别是探索和阐明了Notch信号通路在TA@mDAM三维支架促进BMSCs的成骨分化和促进大鼠颅骨缺损修复过程中的作用和机制。本项目为新型矿化胶原三维骨修复支架的进一步研究和转化应用提供实验基础和理论依据。