Efficient and rapid osteoinduction in an immune-competent host.

Osteoinductive systems to induce targeted rapid bone formation hold clinical promise, but development of technologies for clinical use that must be tested in animal models is often a difficult challenge. We previously demonstrated that implantation of human cells transduced with Ad5F35BMP2 to express high levels of bone morphogenetic protein-2 (BMP2) resulted in rapid bone formation at targeted sites. Inclusion of human cells in this model precluded us from testing this system in an immune-competent animal model, thus limiting information about the efficacy of this approach. Here, for the first time we demonstrate the similarity between BMP2-induced endochondral bone formation in a system using human cells in an immune-incompetent mouse and a murine cell-based BMP2 gene therapy system in immune-competent animals. In both cases the delivery cells are rapidly cleared, within 5 days, and in neither case do they appear to contribute to any of the structures forming in the tissues. Endochondral bone formation progressed through a highly ordered series of stages that were both morphologically and temporally indistinguishable between the two models. Even longterm analysis of the heterotopic bone demonstrated similar bone volumes and the eventual remodeling to form similar structures. The results suggest that the ability of BMP2 to rapidly induce bone formation overrides contributions from either immune status or the nature of delivery cells.

Adenovirus BMP2-induced osteogenesis in combination with collagen carriers.

Adenovirus BMP2 gene therapy has potential of a robust endogenous BMP2 production, while circumventing many of the problems currently associated with recombinant BMP2. The study objective was to determine and compare the ability of adenovirus BMP2 ex vivo gene therapy in combination with three types of collagen carriers to release BMP2 in vitro and to induce heterotopic bone formation in vivo. Human CD45-negative bone marrow cells were ex vivo transduced with a chimeric Ad5F35BMP2. The bioactivity of BMP2 produced by the transduced cells without a carrier, or in combination with three types of collagen carriers (injectable gel, microporous sponge, collagen-mineral composite) was measured and compared to rhBMP2. The heterotopic osteoinductivity assay was performed in immunocompromised NOD/SCID mice. A statistically significant decrease in the amount of rhBMP2 and adenoviral BMP2 released in vitro from the collagen-mineral composite carrier was noted (21% and 12%, respectively), whereas the amounts of rhBMP2 and adenoviral BMP2 released from the gel or sponge carriers were comparable. In vivo, 14 days post-implantation, no bone was formed consistently in groups with the empty Ad5F35HM4 control vector. New bone formation was evident radiographically and histologically in all groups with the Ad5F35BMP2-transduced cells irrespective of the presence or absence of a carrier. The presence of a carrier resulted in osteogenesis limited to the implantation site, and was most pronounced for solid (sponge, composite) carriers. The physical characteristics of the carrier determined the new bone spatial distribution at the site. Solid carriers reduced the clearance of AD5F35-transduced cells by the host immune cells. Adenoviral ex vivo BMP2 gene therapy in combination with collagen carriers with distinct physical characteristics offers the prospects of adjusting this approach to optimally match the specific therapeutic requirements.

Oxygen tension directs chondrogenic differentiation of myelo-monocytic progenitors during endochondral bone formation.

Synthesis of bone requires both essential progenitors to form the various structures and the correct microenvironment for their differentiation. To identify these factors, we have used a system that exploits bone morphogenetic protein's ability to induce endochondral bone formation rapidly. One of the earliest events observed was the influx and proliferation of fibroblastic cells that express both vascular smooth muscle cell markers, alpha smooth muscle actin (alpha SMA), smooth muscle myosin heavy chain, and the monocytic marker CD68. The expression of these factors was lost by days 4 to 5, coincident with the up-regulation of Sox9 and the appearance of chondrocytes. Studies with a cyclization recombination (Cre)/lox system, in which a myeloid-specific promoter driving Cre recombinase can irreversibly unblock expression of beta-galactosidase only in cells of myeloid origin, showed specific activity in the newly formed chondrocytes. These results suggest that early chondrocyte progenitors are of myeloid origin. Simultaneous with this recruitment, we determined that a numbers of these cells were in a hypoxic state, indicative of a low-oxygen environment. The cells in the hypoxic regions were undergoing chondrogenesis, whereas cells in adjacent normoxic regions appeared to be assembling into new vessels, suggesting that the oxygen microenvironment is critical for establishment of the cartilage.

Novel compound enables high-level adenovirus transduction in the absence of an adenovirus-specific receptor.

Viral vectors are extensively used to deliver foreign DNA to cells for applications ranging from basic research to potential clinical therapies. A limiting step in this process is virus uptake and internalization into the target cells, which is mediated by membrane receptors. Although it is possible to modify viral capsid proteins to target the viruses, such procedures are complex and often unsuccessful. Here we present a rapid, inexpensive system for improving transduction of cells, including those that lack receptors for adenovirus fiber proteins. Addition of GeneJammer (Stratagene, La Jolla, CA) during the adenovirus transduction led to a significant increase in both the total number of transduced cells and the level of transgene expression per cell. Studies using cell lines deficient in adenovirus receptors demonstrated that addition of GeneJammer provided a novel cellular entry mechanism for the virus. These findings were tested in a cell-based gene therapy system for the induction of bone, which is contingent on high-level expression of the transgene. Inclusion of GeneJammer in either Ad5BMP2 or Ad5F35BMP2 transduction of a variety of cells demonstrated a correlating increase in bone formation. The results suggest a novel and versatile method for achieving high-level transduction using adenovirus.

Hypoxic adipocytes pattern early heterotopic bone formation.

The factors contributing to heterotopic ossification, the formation of bone in abnormal soft-tissue locations, are beginning to emerge, but little is known about microenvironmental conditions promoting this often devastating disease. Using a murine model in which endochondral bone formation is triggered in muscle by bone morphogenetic protein 2 (BMP2), we studied changes near the site of injection of BMP2-expressing cells. As early as 24 hours later, brown adipocytes began accumulating in the lesional area. These cells stained positively for pimonidazole and therefore generated hypoxic stress within the target tissue, a prerequisite for the differentiation of stem cells to chondrocytes and subsequent heterotopic bone formation. We propose that aberrant expression of BMPs in soft tissue stimulates production of brown adipocytes, which drive the early steps of heterotopic endochondral ossification by lowering oxygen tension in adjacent tissue, creating the correct environment for chondrogenesis. Results in misty gray lean mutant mice not producing brown fat suggest that white adipocytes convert into fat-oxidizing cells when brown adipocytes are unavailable, providing a compensatory mechanism for generation of a hypoxic microenvironment. Manipulation of the transcriptional control of adipocyte fate in local soft-tissue environments may offer a means to prevent or treat development of bone in extraskeletal sites.