激光共聚焦显微镜观测体内回植的GFP标记的3D打印组织工程颌骨

目的:激光共聚焦显微镜观察绿色荧光蛋白(Green Fluorescent Protins,GFP)标记的3D打印组织工程颌骨回植后体内移植情况,为筛选组织工程颌骨并为其后续功能研究提供技术基础。方法:采用羊椎骨煅烧制备陶瓷化骨粉,结合聚乙烯醇制备组织工程颌骨支架,复合GFP慢病毒转染的羊骨髓间充质干细胞(Bone Marrow mesenchymal Stem Cells, BMSCs),回植羊颌骨缺损动物模型,使用激光共聚焦显微镜观察不同时间点其体内分布及存活情况。结果:GFP慢病毒转染的BMSCs,流式细胞仪检测转染率为95.4%,基因测序结果与GFP基因一致。回植后使用激光共聚焦显微镜三维立体成像技术观察到GFP标记的BMSCs在组织工程骨支架上存活3个月以上。结论:激光共聚焦显微镜三维重建技术可很好地观察GFP标记的3D打印组织工程颌骨支架上的细胞移植情况,为组织工程骨的动物实验研究提供了很好的观察方法。

Observation of GFP-labeled 3D-printed Jaw using Laser Confocal Microscopy in Vivo.

Objective: To observe GFP-labeled 3D-printed jaw by using laser confocal microscopy in vivo and to provide technical basis for the follow-up function study. Methods: The calcined ceramic powder was made from sheep vertebrae, which was combined with polyvinyl alcohol to form jaw scaffold. Sheep BMSCs were infected by GFP lentivirus and cultured on the scaffold. The scaffold was implanted into sheep jaw defect and observed by confocal laser scanning microscope at different time points. Results: The infection efficiency was about 95.4% detected by flow cytometry. BMSCs could survive for more than 3 months on the scaffold determined by three-dimensional imaging technique using laser scanning confocal microscopy after implantation. Conclusion: Laser scanning confocal microscopy can be used to observe the GFP-labeled 3D-printed jaw.