新型促血管化海藻酸盐水凝胶骨组织工程支架的构建

Nowadays, owing to insufficient vascularization, the application of bone tissue engineering was limited especially for large bone defect. The structure and biological properties of scaffold are key factors for vascularized bone tissue engineering. In this study, we select photocrosslinked alginate hydrogel as a model. Through adjusting gel structure, degradation rate and biological property, a new type of scaffold with enhanced vascularization is fabricated. After oxidation and photocrosslinked modification, the modified alginate is mixtured with strontium ions which are chelated by DM-nitrophen. A series of scaffolds with large pore size, different gegradation rates or strontium ions contents are prepared and characterized. Following, MSCs are encapsulated into these hydrogels and cultured with osteogenic induce supplements. Then the cell-scaffold constructs are implanted into subcutaneous area or bone defect of femur. The vascularization and osteogenesis of each scaffold are investigated both in vitro and in vivo. The aim of this project is searching for proper parameters of scaffolds to improve vascularized engineered bone formation, to accelerate the clinical translation of scaffold-based bone tissue engineering therapy.

目前，由于血管化不足，骨组织工程在大面积骨缺损修复中仍未取得理想效果。支架材料的结构及生物性能，是组织工程骨血管化的关键因素。本项目采用光交联型海藻酸盐水凝胶作为模型，通过对支架的结构、降解度及生物性能进行改性，制备出大孔径、具备适宜降解度和良好生物活性的新型支架，以促进其内的血管生成。通过对海藻酸钠进行氧化及光照交联基团改性，与包裹锶离子的DM-n载体复合，分别制备具备大孔径、不同降解度或锶离子含量的光交联型海藻酸盐水凝胶支架。将大鼠骨髓基质干细胞包裹至水凝胶中，经体外成骨诱导培养后，分别植入皮下和股骨缺损处，在异位和正位检验血管化及成骨情况，旨在通过优化支架的结构、降解度及生物活性等指标来促进组织工程骨的血管化，解决临床上因肿瘤、炎症、外伤、牙周病等导致的骨缺损修复的难题。

大面积骨缺损的修复一直是临床治疗的难点。本项目拟采用光交联型海藻酸盐水凝胶作为模型，通过氧化改性调整支架的降解度、引入锶离子改善支架的生物性能，制备一种新型支架，以促进其内的血管生成和成骨。我们根据研究方案制备了不同降解度的光交联海藻酸盐水凝胶支架，检测结果显示：四组材料在核磁下均可检测到双键的特征峰，实验组出现不同幅度的氧化峰；扫描电镜下样本均呈疏松多孔的三维纤维网状结构；随着氧化度的提高，水凝胶的溶胀率降低、降解率升高、弹性模量明显降低。由于DM-n载体包裹锶离子成本过高，本研究改用二次交联法将锶离子引入支架中，结果显示：随着锶离子引入量的增加，扫描电镜下支架的孔径呈递减趋势，能谱仪检测到支架中锶元素含量增加、钠元素含量降低，水凝胶的溶胀率和降解速度明显降低，细胞在支架表面的粘附伸展更佳，凝胶内的成骨矿化加强；然而，细胞共培养及三维培养细胞活性及增殖检测显示，过高浓度会对细胞产生一定的抑制效应。此外，本研究增加了光引发剂浓度对光交联海藻酸盐支架性能的影响的探索，结果显示：随着光引发剂浓度的升高，水凝胶中双键交联反应更加完全，扫描电镜下孔径相近，水凝胶的溶胀率减小、降解速度降低、强度增加；LIVE/DEAD测试示各凝胶组内细胞活性良好，最高浓度组细胞活性及增殖均受到轻微抑制。本研究中复合锶离子及光引发剂浓度对光交联海藻酸盐水凝胶支架机械和生物学性能的影响，目前均未见文献报道。