[论文解读] Synthesis, characterization, and biological evaluation of gelatin-based scaffolds

Giuseppe Tronci|arXiv (Cornell University)|Jan 9, 2011

Electrospun Nanofibers in Biomedical Applications被引用 3

一句话总结

本研究采用二异氰酸酯交联法在泡沫体系中合成了共价交联的明胶基支架，实现了可调控的孔隙率（65–73 vol.%）和孔径（117–166 μm）。支架表现出形状恢复能力、可控降解性以及完全的细胞相容性，显示出在骨再生应用中具有良好的机械稳定性与低免疫原性潜力。

ABSTRACT

This thesis presents the development of entropy-elastic gelatin based networks in the form of films or scaffolds. The materials have good prospects for biomedical applications, especially in the context of bone regeneration. Entropy-elastic gelatin based hydrogel films with varying crosslinking densities were prepared with tailored mechanical properties. Gelatin was covalently crosslinked in water above its sol gel transition, which suppressed the gelatin chain helicity. Amorphous films were prepared with tailorable degrees of swelling and wet state Young's modulus. The knowledge gained with this bulk material was transferred to the integrated process of foaming and crosslinking to obtain porous gelatin-based scaffolds. A gelatin solution was foamed in the presence of saponin and the resulting foam was fixed by chemical crosslinking with a diisocyanate. The scaffolds were analyzed in the dry state by micro computed tomography (μCT, porosity: 65\pm 11-73\pm 14 vol.-%), and scanning electron microscopy (SEM, pore size: 117\pm 28-166 \pm 32 μm). After equilibration with water, the scaffolds were form-stable and displayed shape recovery after removal of mechanical loads. The composition dependent compression moduli (Ec: 10 50 kPa) were comparable to the bulk micromechanical Young's moduli, which were measured by atomic force microscopy (AFM). The hydrolytic degradation profile could be adjusted, and a controlled decrease of mechanical properties was observed. The scaffold cytotoxicity and immunologic responses were analyzed in vitro. Indirect eluate tests were carried out with L929 cells so that fully cytocompatible scaffolds were obtained. Furthermore, the material immune response was investigated in vitro. Minimal material endotoxin contamination was successfully achieved (<0.5 EU/mL) by using low-endotoxin gelatin and performing all synthetic steps in cleanroom.

研究动机与目标

• 开发用于生物医学应用（特别是骨再生）的熵弹性明胶基水凝胶网络。
• 通过发泡和化学交联技术，构建具有可控孔隙率和孔径的多孔支架。
• 通过使用低内毒素明胶和无菌环境合成，确保细胞相容性并最小化免疫原性反应。
• 通过调节交联密度来调控机械性能，并评估形状恢复能力和降解特性。
• 建立一种可扩展、生物相容的支架系统，适用于体外和体内测试。

提出的方法

• 在明胶的浊-凝胶转变温度以上，在水溶液中使用二异氰酸酯对明胶进行共价交联，以抑制螺旋结构的形成，从而构建熵弹性网络。
• 采用以皂苷作为表面活性剂的发泡工艺，生成稳定的泡沫前驱体，用于多孔支架的形成。
• 通过化学交联固定泡沫结构，随后进行干燥，并利用显微计算机断层扫描（micro-CT）和扫描电镜（SEM）对孔隙率和形貌进行表征。
• 通过压缩测试和原子力显微镜（AFM）在干燥和湿润状态下测量杨氏模量和压缩模量，以量化机械性能。
• 通过间接浸提液测试（使用L929成纤维细胞）评估细胞毒性，通过内毒素水平分析评估免疫原性。
• 在水性条件下监测降解行为，以评估机械性能随时间的衰减情况。

实验结果

研究问题

• RQ1 在泡沫体系中对明胶进行共价交联是否能够制备出具有可调控孔隙率和孔径、适用于组织工程的多孔支架？
• RQ2 交联密度与机械性能（如杨氏模量和压缩模量）之间存在何种关系？
• RQ3 所制备的支架在水合条件下是否表现出形状恢复能力和结构稳定性？
• RQ4 支架在多大程度上支持细胞活力并表现出最小的免疫原性反应？
• RQ5 是否能够调控支架的降解行为，使其与组织再生的时间进程相匹配？

主要发现

• 通过micro-CT和SEM确认，支架的孔隙率范围为65±11至73±14 vol.%，孔径范围为117±28至166±32 μm。
• 水合后，支架在受力加载后表现出完全的形状恢复和结构稳定性，表明其具有优异的弹性行为。
• 压缩模量（Ec）范围为10至50 kPa，与通过原子力显微镜获得的体积分形微机械测量结果高度一致。
• 支架表现出受控的水解降解行为，机械性能随时间呈渐进性下降。
• 通过L929细胞的间接浸提液测试证实了完全的细胞相容性。
• 内毒素水平成功控制在0.5 EU/mL以下，表明由于使用低内毒素明胶和无菌环境合成，其免疫原性潜力极低。