We report studies of bone tissue engineering using human mesenchymal stem cells (MSCs), a protein substrate (film or scaffold; fast degrading unmodified collagen, or slowly degrading cross-linked collagen and silk), and a bioreactor (static culture, spinner flask, or perfused cartridge). MSCs were isolated from human bone marrow, characterized for the expression of cell surface markers and the ability to undergo chondrogenesis and osteogenesis in vitro, and cultured for 5 weeks. MSCs were positive for CD105/endoglin, and had a potential for chondrogenic and osteogenic differentiation. In static culture, calcium deposition was similar for MSC grown on collagen scaffolds and films. Under medium flow, MSC on collagen scaffolds deposited more calcium and had a higher alcaline phosphatase (AP) activity than MSC on collagen films. The amounts of DNA were markedly higher in constructs based on slowly degrading (modified collagen and silk) scaffolds than on fast degrading (unmodified collagen) scaffolds. In spinner flasks, medium flow around constructs resulted in the formation of bone rods within the peripheral region, that were interconnected and perpendicular to the construct surface, whereas in perfused constructs, individual bone rods oriented in the direction of fluid flow formed throughout the construct volume. These results suggest that osteogenesis in cultured MSC can be modulated by scaffold properties and flow environment.

Abstract-Endothelial vascular adhesion molecule-1 (VCAM-1) is a critical component of the leukocyte-endothelial adhesion cascade, and its strict temporal and spatial regulation make it an ideal target for imaging and therapy. The goal of this study was to develop novel VCAM-1-targeted imaging agents detectable by MRI and fluorescence imaging using phage display-derived peptide sequences and multimodal nanoparticles (NPs). We hypothesized that VCAM-1-mediated cell internalization of phage display-selected peptides could be harnessed as an amplification strategy to chaperone and trap imaging agents inside VCAM-1-expressing cells, thus improving target-to-background ratios. To accomplish our goal, iterative phage display was performed on murine endothelium under physiological flow conditions to identify a family of VCAM-1-mediated cell-internalizing peptides. One specific sequence, containing the VHSPNKK motif that has homology to the ␣-chain of very late antigen (a known ligand for VCAM-1), was shown to bind VCAM-1 and block leukocyte-endothelial interactions. Compared with VCAM-1 monoclonal antibody, the peptide showed 12-fold higher target-to-background ratios. A VHSPNKK-modified magnetofluorescent NP (VNP) showed high affinity for endothelial cells expressing VCAM-1 but surprisingly low affinity for macrophages. In contrast, a control NP without VCAM-1-targeting sequences showed no affinity for endothelial cells. In vivo, VNP successfully identified VCAM-1-expressing endothelial cells in a murine tumor necrosis factor-␣-induced inflammatory model and colocalized with VCAM-1-expressing cells in atherosclerotic lesions present in cholesterol-fed apolipoprotein E apoE Ϫ/Ϫ mice. These results indicate that: (1) small peptide sequences can significantly alter targeting of NPs, (2) the used amplification strategy of internalization results in high target-to-background ratios, and (3)

It has been suggested that vascular cell adhesion molecule-1 (VCAM-1) could serve as an early marker for inflammation of the endothelium. The ability to noninvasively image VCAM-1 could thus be a useful tool to diagnose a number of inflammatory diseases at early stages. Here we demonstrate that magnetooptical nanoparticles conjugated to anti-VCAM-1 antibodies can be used to specifically detect VCAM-1 expression on endothelial cells in culture and in vivo. Elevated VCAM-1 expression was detected on cultured murine heart endothelial cells by both fluorescence and magnetic resonance, while only basal expression levels were detected on murine dermal endothelial cells. Intravital microscopy of a murine inflammatory model injected with the VCAM-1 targeted nanoparticles revealed specific labeling of the activated endothelium, with labeling kinetics yielding a maximum vessel wall signal 6 h after injection. In contrast, nontargeted nanoparticles did not exhibit any specific labeling of the endothelium. These studies suggest that the developed nanoparticle would be useful for MR and optical detection of activated endothelium.