Adenoviral Vector-Mediated Expression of bone morphogenetic Protein-4 in mesenchimal stem cells induces differentiation into the osteoblastic lineage and bone production in vivo

Bone regeneration is critical to the effective management of many bone and muscoloskeletal disorders, such as fracture healing, spinal fusion and osteoporosis. Bone morphogenetic proteins (BMPs) belong to the TGF-beta superfamily and play an important role in embryonic development and bone formation; at least 15 types of BMPs have been identified in humans and several forms of BMPs, mostly rhBMP-2 rhBMP-4 and rhBMP-7, have been shown to induce bone formation in vitro and in vivo. BMPs have also been used in combination with human mesenchymal stem cells (hMSC) that have the capacity to differentiate into bone cell lineages and have generated a great deal of interest because of their potential use in regenerative medicine and tissue engineering. We constructed a human BMP4-expressing first generation adenoviral vector using the CAG promoter for maximal expression of the transgene (FG-AdhBMP4). We purified hMSCs from bone marrow from donors using a physical separation; after two weeks of culture we immuno-depleted the obtained cells using commercially available antibodies against human glycophorin and CD45; we then characterized the resulting cells by citofluorimetry. Immuno-depleted cells were more homogeneous in staining with several markers (CD90, CD13, CD49b) and were negative for HLA-DR, showing only a mild positivity to HLA-ABC. We then infected hMSC cells with the FG-AdhBMP4 vector and observed very high levels of transduction and homogeneous commitment to the osteoblastic lineage. In addition, we purified MSCs from rabbit, transduced them with FG-AdhBMP4 and loaded them onto poly-caprolacton (PCL) scaffolds. The scaffolds were then implanted in a rabbit model of discontinuous bone fracture and lesions healing was compared to controls treated with the scaffolds alone and scaffolds containing untransduced rMSCs. Scaffolds containing rMSCs showed homogeneous formation of bone and reabsorption of PCL by osteoclasts, whereas hystological analysis of bones containing unloaded scaffolds showed areas of vascularization but absence of bone tissue.