Converse Relationship Between In Vitro Osteogenic Differentiation and In Vivo Bone Healing Elicited by Different Populations of Muscle-Derived Cells Genetically Engineered to Express BMP4

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Abstract
In this study, we compared the use of primary muscle‐derived osteoprogenitor cells (PP6 cells) for the delivery of BMP4 to improve bone healing to that of muscle‐derived non‐osteoprogenitor cells (PP1 cells). Surprisingly, the use of PP1 cells resulted in an improved outcome because of the lack of adverse responses to BMP4 involving cell differentiation, proliferation, and apoptosis. Introduction: Although researchers frequently opt to use osteogenic cells for osteogenic bone morphogenetic protein (BMP)‐based ex vivo gene therapy to improve bone healing, it remains unclear whether the osteogenic potential of a cellular vehicle affects the outcome of bone healing applications. Here we compared the use of muscle‐derived non‐osteoprogenitor cells (PP1 cells) to that of primary muscle‐derived osteoprogenitor cells (PP6 cells) for the delivery of BMP4 to improve the healing of bone defects. Materials and Methods: Two distinct populations of primary rat muscle‐derived cells—PP1 and PP6—were selected, transduced with retroviral vectors to express BMP4 or a marker gene (LacZ), and implanted into critical‐sized calvarial defects created in syngeneic rats. The bone healing was monitored radiographically and histologically at 7 and 14 weeks after implantation. Cellular responses to BMP4 were evaluated by alkaline phosphatase histochemical staining and RT‐PCR of another osteogenic marker to indicate osteogenic differentiation, a cell proliferation assay and BrdU (bromodeoxyuridine) labeling to assess cell proliferation, and the TUNEL assay to determine apoptosis. Results and Conclusions: In all animals (nine rats per group), transduced PP1 cells expressing BMP4 demonstrated significantly advanced healing compared with PP6 cells expressing BMP4 and control cells expressing LacZ. We found that constitutive BMP4 expression negatively impacted the in vitro proliferation and in vivo survival rates of PP6 cells, but not PP1 cells. BMP4 exposure also directly inhibited the proliferation and induced the apoptosis of PP6 cells, but not PP1 cells. The impairment in PP6 cell proliferation was directly associated with the osteogenic differentiation of these cells. These results indicate that PP1 cells are better suited than osteoprogenitor cells for use as cellular vehicles to deliver osteogenic BMP4 to improve bone healing and that cellular behavior in response to a particular gene can be used to predict the cells' performance as delivery vehicles in ex vivo gene therapy.