黑磷强化的内固定新型涂层的抗菌与促骨折愈合作用及机制的研究

To promote facture healing and prevent the infection associated with internal fixation are two main missions to treat fracture. The previous research has proved that improvement of functional coating of internal fixation can produce the biological effect to promoting fracture healing, which also will provide a novel technical means to reduce the infection associated with the internal fixation. However, the existing coatings cannot perfectly integrate both anti-microbial and promoting facture healing effect. Recent studies have shown that as a new photothermal conversion material, black phosphorus nanosheets can generate high temperature of about 41-47 ° C after motivation by near infrared ray, induce irreversible ablation of etiological cell by disrupting cell membranes and denatured proteins, and along with slowly degrading drive in situ biominelization to promote bone regeneration. Utilizing the above physicochemical characteristics, the project is going to load black phosphorus nanoplatelets on the surface of hydroxyapatite coated metal internal fixation to form a new composite coating of black phosphorus/hydroxyapatite. In order to verify the effect of the composite coating to ablate bacteria and promote the fracture healing and to explore its mechanism, this project intends to use simulated body fluids and animal fracture models, using physics, imaging, molecular biology and other research methods to evaluate in vitro photothermal conversion characteristics of the composite coating, in vitro antibacterial efficacy, biosafety and the effect to the proliferation and differentiation of bone marrow mesenchymal cells to reveal molecular mechanism of osteogenesis effect, and use animal models to evaluate whether the composite coating has in vivo double effect to ablate bacteria and promote the fracture healing. The research of this project will provide experimental evidence for the application of this novel composite coating of mental internal fixation.

促进骨修复并避免内固定相关感染是骨折治疗的两大任务。前期研究验证内固定功能涂层可产生促进骨修复的作用，也为内固定相关感染的预防提供了新的技术；但现有涂层仍无法有机整合抗菌及促进骨修复的双重功能。黑磷纳米片作为新型的光热转化材料，由红外光激发后产生41-47度的高温，通过破坏细胞膜和变性蛋白诱导不可逆的病原细胞消融，且缓慢降解并启动原位生物矿化过程以促进骨修复。利用以上理化特性，本课题拟将黑磷纳米片加载于羟基磷灰石涂布的内固定表面形成黑磷纳米片/羟基磷灰石的内固定新型涂层。为验证该涂层的抗菌与促进骨修复的双重功能并探讨其机制，本课题拟利用模拟体液及骨折模型等，采用物理学、影像学、分子生物学等研究方法，观察该复合涂层的体外光热转化特性、生物安全性、体外抗菌效能及对骨髓间充质干细胞增值和分化的影响以揭示其成骨效应的细胞机制，并评价体内抗菌及促骨修复的作用。本研究将为该新型涂层的应用提供实验依据。

内植物相关感染严重影响内固定术后骨折愈合的进程。内植物仿生涂层材料体系的不断深入研究为解决以上问题提供了新的思路。二维黑磷纳米片（BPs）作为新型的光热转化材料，由近红外光激发后局部产生高温（42℃），通过破坏细胞膜和变性蛋白质诱导不可逆的病原细胞消融，且缓慢降解后释放出PO3-，其可促进原位生物矿化进而促进骨折修复。在此，课题组首次将BPs加载到涂布有羟基磷灰石（HA）的金属植入物表面，构建新型BPs@HA复合内固定涂层，从而赋予原始内固定清除病原体和促进骨再生的双重生物学功能。进而，利用模拟体液及大鼠骨折模型等，采用物理学、影像学、分子生物学等试验方法验证：BPs@HA复合涂层在消融细菌生物膜和加速骨折愈合方面的优异性能，并表现出良好的生物相容性和生物降解性。此外，通过绝对定量转录组测序、差异表达基因筛选，初步筛选并确定BPs@HA复合涂层调控的与骨形成相关的差异表达基因，并通过功能富集分析，进一步揭示BPs通过代谢相关通路有效调控与BMSCs分化和骨形成相关核心标志物的表达。因此，该研究不仅揭示了BPs@HA复合涂层在消除细菌和加速骨折愈合方面的巨大临床应用潜能，而且有助于深入理解该复合涂层促进成骨生理功能的内在分子机制。