Plastic Surgery Research Council
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Presenter: Michael T Chung
Co-Authors: Morrison SD, Liu C, Zimmermann AS, Paik KJ, Lo DD, Hyun JS, McArdle A, Walmsley G, Senarath-Yapa K, Montoro DT, Sorkin M, Rennert R, Hu M, Chen HH, Chung AS, Longaker MT, Wan DC
Stanford University

Background: Stem cell-based bone engineering has shown great promise as an approach for treating patients with skeletal defects. FACS has identified CD90+ ASCs with enhanced osteogenesis. More recently, inhibition of Noggin (NOG) has been shown to leave endogenously produced BMPs relatively unopposed, also resulting in enhanced osteogenesis. The present study investigated the potential of a combinatorial approach, employing both an isolation of CD90+ ASCs via FACS and modulation of BMP signaling, to optimize bone regeneration.

Methods: Magnet assisted transfection was used to deliver minicircle (MC) NOG shRNA into CD90+ and unsorted cells. These cells were then treated with ODM in vitro. Alkaline phosphatase and quantification were performed on Day 7, alizarin red staining and quantification on Day 14. Osteogenic gene expression was examined by qRT-PCR. For evaluation of in vivo osteogenesis, critical-sized calvarial defects in nude mice were treated with a novel MC-releasing HA-PLGA scaffold prepared using supercritical CO2. MC plasmids were labeled with magnetic nanoparticles, and an external magnet was used to transfect ASCs seeded onto the scaffold. Healing was followed using micro-CT scans for eight weeks. Calvaria were harvested at Week 8, and sections were stained with Movat s Pentachrome.

Results: Transcriptional analysis revealed that the CD90+ ASCs were enriched for a more osteogenic subtype. Suppression of NOG resulted in increased osteogenic gene expression and in vitro osteogenic differentiation, as demonstrated by alkaline phosphatase and alizarin red stainings. In vivo, over the course of eight weeks, the NOG-suppressed CD90+ subpopulation was found to consistently outperform other defects treated with the NOG-suppressed unsorted cells.

Conclusions: Our findings demonstrate that the use of CD90-selected ASCs may facilitate more rapid regeneration of skeletal defects. Furthermore, NOG knockdown may serve to augment bone differentiation through an increase in BMP signaling. The integration of these two strategies may lead to developing promising treatments for clinical translation.

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