Vessel Formation Is Induced Prior to the Appearance of Cartilage in BMP-2-Mediated Heterotopic Ossification

Heterotopic ossification (HO), or endochondral bone formation at nonskeletal sites, often results from traumatic injury and can lead to devastating consequences. Alternatively, the ability to harness this phenomenon would greatly enhance current orthopedic tools for treating segmental bone defects. Thus, understanding the earliest events in this process potentially would allow us to design more targeted therapies to either block or enhance this process. Using a murine model of HO induced by delivery of adenovirus-transduced cells expressing bone morphogenetic protein 2 (BMP-2), we show here that one of the earliest stages in this process is the establishment of new vessels prior to the appearance of cartilage. As early as 48 hours after induction of HO, we observed the appearance of brown adipocytes expressing vascular endothelial growth factors (VEGFs) simultaneous with endothelial progenitor replication. This was determined by using a murine model that possesses the VEGF receptor 2 (Flk1) promoter containing an endothelial cell enhancer driving the expression of nuclear-localized yellow fluorescent protein (YFP). Expression of this marker has been shown previously to correlate with the establishment of new vasculature, and the nuclear localization of YFP expression allowed us to quantify changes in endothelial cell numbers. We found a significant increase in Flk1-H2B::YFP cells in BMP-2-treated animals compared with controls. The increase in endothelial progenitors occurred 3 days prior to the appearance of early cartilage. The data collectively suggest that vascular remodeling and growth may be essential to modify the microenvironment and enable engraftment of the necessary progenitors to form endochondral bone. © 2010 American Society for Bone and Mineral Research

Assessing mechanical integrity of spinal fusion by in situ endochondral osteoinduction in the murine model

<p>Abstract</p> <p>Background</p> <p>Historically, radiographs, micro-computed tomography (micro-CT) exams, palpation and histology have been used to assess fusions in a mouse spine. The objective of this study was to develop a faster, cheaper, reproducible test to directly quantify the mechanical integrity of spinal fusions in mice.</p> <p>Methods</p> <p>Fusions were induced in ten mice spine using a previously described technique of in situ endochondral ossification, harvested with soft tissue, and cast in radiolucent alginate material for handling. Using a validated software package and a customized mechanical apparatus that flexed and extended the spinal column, the amount of intervertebral motion between adjacent vertebral discs was determined with static flexed and extended lateral spine radiographs. Micro-CT images of the same were also blindly reviewed for fusion.</p> <p>Results</p> <p>Mean intervertebral motion between control, non-fused, spinal vertebral discs was 6.1 ± 0.2° during spine flexion/extension. In fusion samples, adjacent vertebrae with less than 3.5° intervertebral motion had fusions documented by micro-CT inspection.</p> <p>Conclusions</p> <p>Measuring the amount of intervertebral rotation between vertebrae during spine flexion/extension is a relatively simple, cheap (<$100), clinically relevant, and fast test for assessing the mechanical success of spinal fusion in mice that compared favorably to the standard, micro-CT.</p

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 upregulation 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

Hydrogel Microsphere Encapsulation of a Cell-Based Gene Therapy System Increases Cell Survival of Injected Cells, Transgene Expression, and Bone Volume in a Model of Heterotopic Ossification

Bone morphogenetic proteins (BMPs) are well known for their osteoinductive activity, yet harnessing this capacity remains a high-priority research focus. We present a novel technology that delivers high BMP-2 levels at targeted locations for rapid endochondral bone formation, enhancing our preexisting cell-based gene therapy system by microencapsulating adenovirus-transduced cells in nondegradable poly(ethylene glycol) diacrylate (PEGDA) hydrogels before intramuscular delivery. This study evaluates the in vitro and in vivo viability, gene expression, and bone formation from transgenic fibroblasts encapsulated in PEGDA microspheres. Fluorescent viability and cytotoxicity assays demonstrated >95% viability in microencapsulated cells. ELISA and alkaline phosphatase assays established that BMP-2 secretion and specific activity from microencapsulated AdBMP2-transduced fibroblasts were not statistically different from monolayer. Longitudinal transgene expression studies of AdDsRed-transduced fibroblasts, followed through live animal optical fluorescent imaging, showed that microencapsulated cells expressed longer than unencapsulated cells. When comparable numbers of microencapsulated AdBMP2-transduced cells were intramuscularly injected into mice, microcomputed tomography evaluation demonstrated that the resultant heterotopic bone formation was approximately twice the volume of unencapsulated cells. The data suggest that microencapsulation protects cells and prolongs and spatially distributes transgene expression. Thus, incorporation of PEGDA hydrogels significantly advances current gene therapy bone repair approaches