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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.

Decellularization is a promising new method to prepare natural matrices for tissue regeneration. Successful decellularization has been reported using various tissues including skin, tendon, and cartilage, though studies using hard tissue such as bone are lacking. In this study, we aimed to define the optimal experimental parameters to decellularize natural bone matrix using 0.5% sodium dodecyl sulfate and 0.1% NH 4 OH. Then, the effects of decellularized bone matrix on rat mesenchymal stem cell proliferation, osteogenic gene expression, and osteogenic differentiations in a two-dimensional culture system were investigated. Decellularized bone was also evaluated with regard to cytotoxicity, biochemical, and mechanical characteristics in vitro. Evidence of complete decellularization was shown through hematoxylin and eosin staining and DNA measurements. Decellularized bone matrix displayed a cytocompatible property, conserved structure, mechanical strength, and mineral content comparable to natural bone. To study new bone formation, implantation of decellularized bone matrix particles seeded with rat mesenchymal stem cells was conducted using an orthotopic in vivo model. After 3 months post-implantation into a critical-sized defect in rat calvaria, new bone was formed around decellularized bone matrix particles and also merged with new bone between decellularized bone matrix particles. New bone formation was analyzed with micro computed tomography, mineral apposition rate, and histomorphometry. Decellularized bone matrix stimulated mesenchymal stem cell proliferation and osteogenic differentiation in vitro and in vivo, achieving effective bone regeneration and thereby serving as a promising biological bone graft.