Marrow Stromal Cell-Based Cyclooxygenase 2 Ex Vivo Gene-Transfer Strategy Surprisingly Lacks Bone-Regeneration Effects and Suppresses the Bone-Regeneration Action of Bone Morphogenetic Protein 4 in a Mouse Critical-Sized Calvarial Defect Model

Abstract

This study evaluated whether the murine leukemia virus (MLV)-based cyclooxygenase-2 (Cox-2) ex vivo gene-transfer strategy promotes healing of calvarial defects and/or synergistically enhances bone morphogenetic protein (BMP) 4-mediated bone regeneration. Gelatin scaffolds impregnated with mouse marrow stromal cells (MSCs) transduced with MLV-expressing BMP4, Cox-2, or a control gene were implanted into mouse calvarial defects. Bone regeneration was assessed by X-ray, dual-energy X-ray absorptiometry, and histology. In vitro, Cox-2 or prostanglandin E(2) enhanced synergistically the osteoblastic differentiation action of BMP4 in mouse MSCs. In vivo, implantation of BMP4-expressing MSCs yielded massive bone regeneration in calvarial defects after 2 weeks, but the Cox-2 strategy surprisingly did not promote bone regeneration even after 4 weeks. Staining for alkaline phosphatase (ALP)-expressing osteoblasts was strong throughout the defect of animals receiving BMP2/4-expressing cells, but defects receiving Cox-2-expressing cells displayed weak ALP staining along the edge of original intact bone, indicating that the Cox-2 strategy lacked bone-regeneration effects. The Cox-2 strategy not only lacked bone-regeneration effects but also suppressed the BMP4-induced bone regeneration. In vitro coculture of Cox-2-expressing MSCs with BMP4-expressing MSCs in gelatin scaffolds reduced BMP4 mRNA transcript levels, suggesting that Cox-2 may promote BMP4 gene silencing in BMP4-expressing cells, which may play a role in the suppressive action of Cox-2 on BMP4-mediated bone formation. In summary, the Cox-2 ex vivo gene-transfer strategy not only lacks bone-regeneration effects but also suppresses the bone-regeneration action of BMP4 in healing of calvarial defects.