力学刺激下HA/PU复合组织工程骨的构建及其生物学响应机制研究

Bone tissue is always affected by mechanical factors in physiological state. The biological mechanism of mechanical stimulation induced function of tissue-engineered bone has not been well defined yet. The natural renewable carbohydrates as the soft segment were chosen to polymerize with aliphatic isocyanate meanwhile mixing with nano-hydroxyapatite to eventually fabricate three-dimensional porous scaffolds. By adjusting the proportion of soft and hard segments or the content of inorganic components, the biodegradable repair materials which possess the appropriate physical and chemical properties, mechanical properties matching with natural bone tissue were obtained. Marrow mesenchymal stem cells (MSCs) were isolated and induced into osteogenic differentiation. In order to construct tissue-engineered bone (TEB), cells were seeded on the scaffolds. The low-intensity pulsed ultrasound (LIPUS) was applied to stimulate the TEB, the different ultrasonic parameters such as intensity, frequency and time were set. The proliferation and viability of seeded cells were detected at celluar level, the expression of osteogenic genes and protein secretion were investigated at the molecular level. To evaluate the effect of bone repair, the bone defect of animals was created, and TEB was implanted into defect zone under LIPUS treatment. The main aims of this study were to explore the optimal mechanical stimulus loading conditions, establish the optimal mechanic microenvironment for tissue regeneration, elucidate the biological response mechanism of TEB stimulated by mechanical signal. This study has great theoretical significance for bone regeneration and reconstruction, and the TEB can be promising candidate for clinical application.

骨组织在生理状态下始终受到力学因素的影响。力学刺激诱导组织工程骨实现功能的生物学机制尚需深入阐明。本项目提出利用天然可再生多糖类高分子为软段、脂肪族异氰酸酯为硬段合成生物质基聚氨酯，并进一步原位复合无机纳米羟基磷灰石制备三维多孔支架。调整软硬段配比及无机组分含量以获得理化性能适宜、力学性能与天然骨组织相匹配的生物降解型修复材料。提取骨髓基质干细胞，定向诱导成骨分化后接种于多孔支架体外构建组织工程骨。加载低强度脉冲超声力学刺激，调节超声强度、频率、时间等参数，在细胞水平检测种子细胞增殖及细胞活力的变化；在分子水平考察细胞成骨相关基因表达及蛋白分泌的情况；建立骨缺损模型，在动物实验水平评价验证超声刺激下该新型组织工程骨植入后的修复重建效果；探索最佳的力学刺激加载条件，建立最优化的组织再生力学微环境，阐明组织工程骨感受力学信号的生物学响应机制。本项目对骨组织再生与重建具有理论意义和临床应用价值。

骨组织在生理状态下始终受到力学因素的影响。力学刺激诱导组织工程骨实现功能的生物学机制尚需深入阐明。本项目提出利用天然可再生多糖类高分子为软段、脂肪族异氰酸酯为硬段合成生物质基聚氨酯，并进一步原位复合无机粒子制备三维多孔支架。调整软硬段配比及无机组分含量以获得理化性能适宜、力学性能与天然骨组织相匹配的生物降解型修复材料。复合多孔支架材料通过红外光谱、X-射线衍射、扫描电子显微镜进行表征。采用液体替代法计算支架的孔隙率。测定了支架的水接触角。支架的力学性能通过单调压缩和循环压缩实验进行测试。结果表明，无机粒子与聚氨酯完全整合成复合生物材料，复合多孔支架材料最大抗压强度和弹性模量分别为5.2 MPa和14.1 MPa，孔隙率最大为73%，最小水接触角为89.16°。复合多孔支架具有良好的回弹性，循环压缩实验表明撤去载荷1小时后支架可以恢复到初始形状的90%左右。评价了支架的体外细胞相容性，结果显示复合多孔支架具有良好的细胞亲和性。构建组织工程骨，加载低强度脉冲超声力学刺激，调节超声强度、频率、时间等参数，检测细胞增殖、细胞活力的变化以及细胞分化的情况。结果表明适宜的复合多孔支架材料联合LIPUS刺激对细胞的增殖和分化可产生直接显著的作用。建立骨缺损模型选用SD大鼠开展动物实验，评价组织工程骨植入后的修复重建效果。结果显示复合多孔支架与宿主骨亲和性好并不产生免疫排斥反应，骨修复效果良好。本项目对骨组织再生与重建具有理论意义和临床应用价值。