装载SDF-1/BMP-2纳米粒及DFM的可注射温敏水凝胶体系对脊柱融合的应用基础研究

Currently, bone grafting fusion cannot achieve satisfactory and effective outcome only relying on bone graft repairing in the field of spine. Vascularization and inducing osteogenesis play the most important roles in bone repairing and reconstruction. Increasing fusion rate with the help of artificial spinal scaffold materials has become a vital spinal fusion research project in the world. In the current project, we combine irregularity and complexity of fusion interface with good fluidity of hydrogel as well as temperature sensitivity of sol-gel transition to construct a composite system, which can inject temperature-sensitive hydrogel. In the hydrogel, the DFM is loaded in, suppressing the degradation of PHD of Hif-1α and increasing the expression level of Hif-1α, promoting local vascularization. On the other hand, the hydrogel time-sequently releases SDF-1 which can be controlled release along with BMP-2 via SDF-1/CXCR-4 signal pathways. In this way, it can promote MSCs homing in, migration and osteoinduction at fusion area in low oxygen conditions to accelerate bone tissue reconstruction and regeneration. We aim to comprehensively study the physical and chemical properties, biocompatibility, chemotaxis of stem cells, induction differentiation, degradation and fusion of the composite system.

脊柱目前单纯依靠植骨修复可能无法达到满意且有效的植骨融合。血管化和诱导成骨是骨修复过程中的两个最重要的环节。通过人工脊柱支架材料提高脊柱融合率，已成为当前国内外脊柱融合研究的重要课题。本项目拟从脊柱融合损伤界面的不规则性及复杂性出发，结合水凝胶良好的流动性、温度敏感溶胶-凝胶转变行为，并利用水凝胶体系对药物及相关因子时序性释放的能力，构建具有的可注射温度敏感水凝胶复合体系。在水凝胶中合理引入对局部血管化具有促进作用的成分DFM，通过首先释放DFM，抑制降解 Hif-1α的酶 PHD，增加局部Hif-1α的有效表达，以持续促进局部血管再生和生成；时序性释放SDF-1，与BMP-2联合控释通过SDF-1/CXCR-4 信号通路,促进MSCs在低氧的脊柱融合部位归巢、迁移和诱导成骨，以促进骨组织的整体修复与再生。我们拟全面研究该体系的理化性质、生物相容性、干细胞趋化、诱导分化、降解及融合等。

本课题拟通过构建具有时序性缓释特性的负载SDF-1/BMP-2纳米粒及DFM的可注射温敏水凝胶体系，探究其在脊柱后路不规则骨缺损中的促血管化、促成骨效果。首先，我们构建了可注射温敏性PLEL水凝胶复合体系。通过不同比例之间的反复调试、检测，最终筛选出复合体系各成分浓度为20wt%PLEL+5wt%nHAP+1wt%COL，通过流变特性、体外细胞实验等，验证了该体系的安全性及有效性。其次，我们构建了负载SDF-1/BMP-2的PCEC纳米粒体系，通过表征、粒径、电位、载药等检测，验证了其稳定性。再次，我们通过体外释放特性、体内促血管化及促成骨效果等探究了DFM的使用剂量，结果显示400μM DFM效果最佳，200μM DFM次之。最后，综合考虑溶解度和成骨效果，我们构建了本课题所需的负载SDF-1/BMP-2纳米粒及DFM的可注射温敏水凝胶体系，各成分浓度及比例为：（20wt%PLEL+5wt%nHAP+1wt%COL+200μM DFM）：SDF-1/BMP-2@PCEC =3：1，其中PLEL水凝胶最终浓度为15wt%，DFM最终浓度150μM/ml，SDF-1最终浓度为1μg/ml，BMP-2最终浓度为1μg/ml。通过检测，验证了该体系具有37℃成胶的温敏性；依次释放DFM、SDF-1和BMP-2的时序性；体外促进细胞增殖、体内促进腰椎后路不规则骨缺损模型血管化及成骨等效果。总的来说，本课题按原计划构建了可注射、温敏性、能够负载不同因子及药物、具有时序性释放特性的水凝胶复合体系，通过材料特性、体外实验、体内实验等验证了该体系的安全性及有效性，能够为脊柱融合提供新的解决思路。

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