促血管再生的中药组方靶向骨修复模式材料的构建与应用基础研究

It is the fact that bone defects and nonunion of bone caused by various reasons hinders the development of clinical treatment for bone, no matter adopting the methods of bone grafting or tissue engineering bone for treatment, "vascularization" remains the urgent issue in the bone healing process. Based on our primary researching work, and guided by the therapies of promoting blood circulation and removing blood stasis as well as reinforcing kidney to strengthen bones, salvia-icariin scaffold for bone repairing was well prepared. In order to explore the possibilities of whether salvia could be served as an angiogenic inducer or not, we inspect its effects on EPCs and BMSCs proliferation and differentiation into vascular endothelial cells. Meanwhile, we construct salvia-icariin bone repair scaffolds which were prepared by the spray drying, in situ hybridization and freeze drying methods. Subsequently, we further examine the physical and chemical characteristics, mechanical properties and biological compatibility of the composite scaffolds to assess the drug loading process, pattern and dosage. Besides, we also test the release behavior and pharmacokinetics of salvia in composite scaffolds in vitro. Finally, we implant the composite scaffolds to estimate the ectopic and in situ bone repair capacities in vivo. This research will provide a beneficial exploration of Chinese herbal compound for tissue engineering, and also extend a new thought for traditional Chinese medicines targeted therapy on traumatic bone diseases.

由各种原因引起的骨缺损、骨不连一直是临床治疗中亟待解决的难题，不论是采用骨移植还是组织工程骨进行治疗，"血管再生"依旧是骨愈合过程所需解决的首要问题。本课题立足于前期工作基础，以中医"活血化瘀、补肾健骨"理论为指导，选取丹参和淫羊藿苷为模型中药，构建丹参－淫羊藿苷骨修复材料。通过考察丹参对内皮祖细(EPCs）和骨髓间充质干细胞(BMSCs)增殖和向血管内皮细胞分化的影响、明确其作用机制，探讨丹参作为诱导血管新生活性因子的可行性；经喷雾干燥、原位复合和冷冻干燥等方法制备丹参－淫羊藿苷骨修复材料，通过考察复合材料的理化特征、力学性能和生物学相容性，评估载药过程、方式、剂量对材料的影响；通过体外释药试验评估材料中丹参的释药行为和释药动力学；通过体内植入试验，评估该组方材料的异位和原位骨修复能力。以期为中药组方应用于组织工程研究提供有益探索，为中医药靶向治疗骨伤疾病提供一条新的思路。

背景：“活血化瘀、补肾健骨”是中医骨伤三期辩证的治法原则。从基础和临床研究来看，其物质基础即为重建骨折部位的微循环，加速血管再生。基于前期工作，课题组采用丹参素和淫羊藿苷构建了中药组方型骨修复材料（tan-ica-CS/HA），并对一系列问题进行了探讨。目的：研究丹参素对hEPCs增殖和分化的影响，评价丹参素作为一种新型血管诱导信号因子应用于骨组织工程的可行性；构建丹参素-淫羊藿苷-CS/HA骨修复材料（tan-ica-CS/HA），探讨其理化性质和生物相容性；研究其的体外释药行为；明确其体内异位成骨和原位骨缺损修复效能。方法：经细胞毒性试验、增殖试验，明确丹参素的细胞毒性范围和评估丹参素对hEPCs增殖的影响；采用管腔形成试验、表面标志物检测，评估丹参素对hEPCs分化的影响。原位复合法制备tan-ica-CS/HA材料，观察材料形貌、分析其化学构成、测试力学性能；评估材料生物学相容性。采用UPLC测试材料中丹参素的释药行为。开展体内试验，评估tan-ica-CS/HA的异位成骨能力和原位骨修复效能。结果：高于10-5M丹参素具有轻度细胞毒性。10-8M丹参素可明显促进EPCs的增殖，缩短细胞周期。10-6M和10-5M丹参素可促进hEPCs向成熟血管内皮细胞分化。原位复合法构建的tan-ica-CS/HA具有良好的孔隙率和孔径，随载药剂量的增加，材料的力学性能降低。FTIR分析表明材料制备过程未损伤丹参素与淫羊藿苷的官能机团。虽然tan-ica-CS/HA不同程度地降低了hEPCs 的增殖活性，但是血液相容性和热原试验检测合格。UPLC检测表明，丹参素在1-3d呈爆发性地从材料中释放，随载丹参素剂量的不同，释放时间维持在15d-30d不等。异位成骨实验表明，tan-ica-CS/HA可提高组织内VEGF和BMP-2表达；组织学观察可见材料内有新生的骨与软骨组织形成；骨密度分析可见软组织内骨矿含量增加。原位成骨实验证实tan-ica-CS/HA可提高骨缺损部位的早期成骨活性，缩短髓腔沟通时间，增加骨矿含量，具有良好的骨修复效能。结论：丹参素可促进hEPCs的增殖和分化；tan-ica-CS/HA具有良好的空间结构和生物学相容性，制备过程对所载中药无损害；tan-ica-CS/HA可在筋膜内诱导产生骨软骨组织，植入骨缺损部位可促进成骨，加速骨修复。