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Use of Novel BMP-2 Minicircle Plasmid-Releasing Scaffolds for Bone Engineering
Michael T. Chung, B.S.1, Kevin J. Paik, A.B.1, Adrian McArdle, M.D.1, Michael S. Hu, M.D.1, Kshemendra Senarath-Yapa, M.D.1, Shane D. Morrison, M.S.1, Graham Walmsley, B.S.1, Ruth Tevlin, M.D.1, Elizabeth Zielins, M.D.1, David Atashroo, M.D.1, Wan Xing Hong, M.S.1, Christopher Duldulao, B.S.1, Taylor Wearda, B.S.1, Rebecca M. Garza, M.D.1, Arash Momeni, M.D.1, Michael Keeney, Ph.D.2, Fan Yang, Ph.D.2, Michael T. Longaker, M.D., M.B.A.1, Derrick C. Wan, M.D.1.
1Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery Division, Stanford, CA, USA, 2Stem Cell and Biomaterials Engineering Laboratory, Stanford, CA, USA.
Skeletal defects are common problems and are difficult to heal using current therapies. Bone morphogenetic protein (BMP)-2 has very strong osteoconductive activity, however, transient exposure to BMP-2 may not be sufficient to stimulate and sustain adequate bone growth for large defects. A gene-therapy approach to BMP manipulation may offer an alternative strategy, but considering the safety concerns associated with viral vectors, a non-integrating technology would be a safer route to enhance BMP signaling. The present study evaluated the potential of a novel BMP-2 minicircle (mc)-releasing scaffold to promote bone regeneration.
A novel mc-releasing scaffold was prepared using supercritical CO2 (scCO2). Luciferase mc was lyophilized with 10% hydroxyapatite (HA) powder and pulverized PLGA. A single-step scCO2 foaming process was utilized to generate porous scaffolds. This scCO2 foaming process was carried out at high pressure (2000 psi) for 1 hour at 35°C. In vitro release assays were performed to determine the release kinetics of Luc-mc from the HA-PLGA scaffolds. The amount of DNA released at each time interval was determined by the PicoGreen® fluorometric assay. To evaluate the time course of mc DNA expression, Luc-mc-releasing scaffolds were implanted into critical-sized (4-mm) calvarial defects in nude mice. To assess minicircle plasmid delivery efficiency, transgene expression was analyzed by in vivo bioluminescence imaging (IVIS). To assess BMP-2 mc’s effect on osteogenesis in vitro, parietal osteoblasts from C57BL/6 mice were transfected with BMP-2 mc and cultured in ODM. At Day 14, alizarin red staining was performed and osteogenic gene expression was measured by qRT-PCR. For evaluation of in vivo osteogenesis, critical-sized (4-mm) calvarial defects in nude mice were treated with a BMP-2 mc-releasing scaffold. Healing was followed using micro-CT scans for eight weeks, and sections were stained with Movat’s Pentachrome and aniline blue.
The scCO2 gas foaming process had little effect on the release profile or DNA integrity. Scaffolds formed with gas foaming had a sustained release of plasmid for at least six weeks with retention of DNA integrity. For Luc-mc-releasing scaffolds, sustained transgene expression was observed for at least eight weeks. Parietal osteoblasts treated with BMP-2 mc exhibited enhanced osteogenesis and mineralization compared to cells treated with a GFP mc control. Over the course of eight weeks, defects treated with the BMP-2 mc-releasing scaffolds were found to consistently outperform defects treated with scaffolds alone.
In summary, porous HA-PLGA scaffolds releasing plasmid DNA induced prolonged in vivo transgene expression up to 8 weeks, which was sufficient to promote physiological response. BMP-2 mc led to increased osteogenic capacity in vitro and scaffold-based delivery of BMP-2 mc facilitated more rapid regeneration of critical-sized calvarial defects.
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