Wang Lufei, Lee Dong Joon, Han Han, Zhao Lixing, Tsukamoto Hiroshi, Kim Yong-Il, Musicant Adele M, Parag-Sharma Kshitij, Hu Xiangxiang, Tseng Henry C, Chi Jen-Tsan, Wang Zhengyan, Amelio Antonio L, Ko Ching-Chang
Division of Oral and Craniofacial Health Sciences, University of North Carolina Adams School of Dentistry, Chapel Hill, NC, USA.
State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Department of Orthodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, China.
J Tissue Eng. 2021 Feb 16;12:2041731421995465. doi: 10.1177/2041731421995465. eCollection 2021 Jan-Dec.
Bioluminescent imaging (BLI) has emerged as a popular in vivo tracking modality in bone regeneration studies stemming from its clear advantages: non-invasive, real-time, and inexpensive. We recently adopted bioluminescence resonance energy transfer (BRET) principle to improve BLI cell tracking and generated the brightest bioluminescent signal known to date, which thus enables more sensitive real-time cell tracking at deep tissue level. In the present study, we brought BRET-based cell tracking strategy into the field of bone tissue engineering for the first time. We labeled rat mesenchymal stem cells (rMSCs) with our in-house BRET-based GpNLuc reporter and evaluated the cell tracking efficacy both in vitro and in vivo. In scaffold-free spheroid 3D culture system, using BRET-based GpNLuc labeling resulted in significantly better correlation to cell numbers than a fluorescence based approach. In scaffold-based 3D culture system, GpNLuc-rMSCs displayed robust bioluminescence signals with minimal background noise. Furthermore, a tight correlation between BLI signal and cell number highlighted the robust reliability of using BRET-based BLI. In calvarial critical sized defect model, robust signal and the consistency in cell survival evaluation collectively supported BRET-based GpNLuc labeling as a reliable approach for non-invasively tracking MSC. In summary, BRET-based GpNLuc labeling is a robust, reliable, and inexpensive real-time cell tracking method, which offers a promising direction for the technological innovation of BLI and even non-invasive tracking systems, in the field of bone tissue engineering.
生物发光成像（BLI）因其具有非侵入性、实时性和低成本等明显优势，已成为骨再生研究中一种流行的体内追踪方式。我们最近采用生物发光共振能量转移（BRET）原理来改进BLI细胞追踪，并产生了迄今为止已知最亮的生物发光信号，从而能够在深部组织水平进行更灵敏的实时细胞追踪。在本研究中，我们首次将基于BRET的细胞追踪策略引入骨组织工程领域。我们用我们内部基于BRET的GpNLuc报告基因标记大鼠间充质干细胞（rMSCs），并在体外和体内评估细胞追踪效果。在无支架球体3D培养系统中，与基于荧光的方法相比，使用基于BRET的GpNLuc标记与细胞数量的相关性显著更好。在基于支架的3D培养系统中，GpNLuc-rMSCs显示出强大的生物发光信号，背景噪声最小。此外，BLI信号与细胞数量之间的紧密相关性突出了使用基于BRET的BLI的强大可靠性。在颅骨临界尺寸缺损模型中，强大的信号以及细胞存活评估的一致性共同支持基于BRET的GpNLuc标记作为一种可靠的非侵入性追踪MSC的方法。总之，基于BRET的GpNLuc标记是一种强大、可靠且低成本的实时细胞追踪方法，为骨组织工程领域的BLI乃至非侵入性追踪系统的技术创新提供了一个有前景的方向。
