Osteogenic preconditioning in perfusion bioreactors improves vascularization and bone formation by human bone marrow aspirates.

Cell-derived extracellular matrix (ECM) provides a niche to promote osteogenic differentiation, cell adhesion, survival, and trophic factor secretion. To determine whether osteogenic preconditioning would improve the bone-forming potential of unfractionated bone marrow aspirate (BMA), we perfused cells on ECM-coated scaffolds to generate naïve and preconditioned constructs, respectively. The composition of cells selected from BMA was distinct on each scaffold. Naïve constructs exhibited robust proangiogenic potential in vitro, while preconditioned scaffolds contained more mesenchymal stem/stromal cells (MSCs) and endothelial cells (ECs) and exhibited an osteogenic phenotype. Upon implantation into an orthotopic calvarial defect, BMA-derived ECs were present in vessels in preconditioned implants, resulting in robust perfusion and greater vessel density over the first 14 days compared to naïve implants. After 10 weeks, human ECs and differentiated MSCs were detected in de novo tissues derived from naïve and preconditioned scaffolds. These results demonstrate that bioreactor-based preconditioning augments the bone-forming potential of BMA.

Altered bone development in a mouse model of peripheral sensory nerve inactivation.

OBJECTIVES: The present study sought to determine the effects of decreased peripheral sensory nerve function on skeletal development and bone metabolism in mice. METHODS: C57BL/6 neonatal mice were treated with capsaicin to induce peripheral sensory nerve degeneration, and compared to vehicle-treated controls at 4, 8 and 12 weeks of age. Changes in bone structure were assessed using micro-computed tomography, mechanical properties and fracture resistance were assessed using three-point bending of radii, and bone turnover was assessed using dynamic histomorphometry and serum biomarkers. RESULTS: Capsaicin treatment resulted in small but significant decreases in bone structure, particularly affecting trabecular bone. Capsaicin-treated mice exhibited lower trabecular thickness at the femoral metaphysis and L5 vertebral body compared with vehicle-treated mice. However, capsaicin- and vehicle-treated mice had similar mechanical properties and bone turnover rates. CONCLUSION: Neonatal capsaicin treatment affected trabecular bone during development; however these small changes may not be meaningful with respect to bone strength under normal loading conditions. It is possible that capsaicin-sensitive neurons may be more important for bone under stress conditions such as increased mechanical loading or injury. Future studies will investigate this potential role of peripheral sensory nerves in bone adaptation.

Neutrophils exposed to A. phagocytophilum under shear stress fail to fully activate, polarize, and transmigrate across inflamed endothelium.

Anaplasma phagocytophilum is an obligate intracellular bacterium that has evolved mechanisms to hijack polymorphonuclear neutrophil (PMN) receptors and signaling pathways to bind, infect, and multiply within the host cell. E-selectin is upregulated during inflammation and is a requisite endothelial receptor that supports PMN capture, rolling, and activation of integrin-mediated arrest. Ligands expressed by PMN that mediate binding to endothelium via E-selectin include sialyl Lewis x (sLe(x))-expressing ligands such as P-selectin glycoprotein ligand-1 (PSGL-1) and other glycolipids and glycoproteins. As A. phagocytophilum is capable of binding to sLe(x)-expressing ligands expressed on PMN, we hypothesized that acute bacterial adhesion to PMN would subsequently attenuate PMN recruitment during inflammation. We assessed the dynamics of PMN recruitment and migration under shear flow in the presence of a wild-type strain of A. phagocytophilum and compared it with a strain of bacteria that binds to PMN independent of PSGL-1. Acute bacterial engagement with PMN resulted in transient PMN arrest and minimal PMN polarization. Although the wild-type pathogen also signaled activation of beta2 integrins and elicited a mild intracellular calcium flux, downstream signals including PMN transmigration and phosphorylation of p38 mitogen-activated protein kinase (MAPK) were inhibited. The mutant strain bound less well to PMN and failed to activate beta2 integrins and induce a calcium flux but did result in decreased PMN arrest and polarization that may have been partially mediated by a suppression of p38 MAPK activation. This model suggests that A. phagocytophilum binding to PMN under shear flow during recruitment to inflamed endothelium interferes with normal tethering via E-selectin and navigational signaling of transendothelial migration.

Annexin V disruption impairs mechanically induced calcium signaling in osteoblastic cells.

The mechanical environment of the skeleton plays an important role in the establishment and maintenance of structurally competent bone. Biophysical signals induced by mechanical loading elicit a variety of cellular responses in bone cells, however, little is known about the underlying mechanotransduction mechanism. We hypothesized that bone cells detect and transduce biophysical signals into biological responses via a mechanism requiring annexin V (AnxV). AnxV, a calcium-dependent phospholipid binding protein, has several attributes, which suggest it is ideally suited for a role as a mechanosensor, possibly a mechanosensitive ion channel. These include the ability to function as a Ca2+ selective ion channel, and the ability to interact with both extracellular matrix proteins and cytoskeletal elements. To test the hypothesis that AnxV has a role in mechanosensing, we studied the response of osteoblastic cells to oscillating fluid flow, a physiologically relevant physical signal in bone, in the presence and absence of AnxV inhibitors. In addition, we investigated the effects of oscillating flow on the cellular location of AnxV. Oscillating fluid flow increased both [Ca2+]i levels and c-fos protein levels in osteoblasts. Disruption of AnxV with blocking antibodies or a pharmacological inhibitor, K201 (JTV-519), significantly inhibited both responses. Additionally, our data show that the cellular location of AnxV was modulated by oscillating fluid flow. Exposure to oscillating fluid flow resulted in a significant increase in AnxV at both the cell and nuclear membranes. In summary, our data suggest that AnxV mediates flow-induced Ca2+ signaling in osteoblastic cells. These data support the idea of AnxV as a Ca2+ channel, or a component of the signaling pathway, in the mechanism by which mechanical signals are transduced into cellular responses in the osteoblast. Furthermore, the presence of a highly mobile pool of AnxV may provide cells with a powerful mechanism by which cellular responses to mechanical loading might be amplified and regulated.