Human perivascular stem cell-derived extracellular vesicles mediate bone repair.

The vascular wall is a source of progenitor cells that are able to induce skeletal repair, primarily by paracrine mechanisms. Here, the paracrine role of extracellular vesicles (EVs) in bone healing was investigated. First, purified human perivascular stem cells (PSCs) were observed to induce mitogenic, pro-migratory, and pro-osteogenic effects on osteoprogenitor cells while in non-contact co-culture via elaboration of EVs. PSC-derived EVs shared mitogenic, pro-migratory, and pro-osteogenic properties of their parent cell. PSC-EV effects were dependent on surface-associated tetraspanins, as demonstrated by EV trypsinization, or neutralizing antibodies for CD9 or CD81. Moreover, shRNA knockdown in recipient cells demonstrated requirement for the CD9/CD81 binding partners IGSF8 and PTGFRN for EV bioactivity. Finally, PSC-EVs stimulated bone repair, and did so via stimulation of skeletal cell proliferation, migration, and osteodifferentiation. In sum, PSC-EVs mediate the same tissue repair effects of perivascular stem cells, and represent an 'off-the-shelf' alternative for bone tissue regeneration.

Frontal Bone Healing Is Sensitive to Wnt Signaling Inhibition via Lentiviral-Encoded Beta-Catenin Short Hairpin RNA.

The Wnt/ß-catenin signaling pathway plays an integral role in skeletal biology, spanning from embryonic skeletal patterning through bone maintenance and bone repair. Most experimental methods to antagonize Wnt signaling in vivo are either systemic or transient, including genetic approaches, use of small-molecule inhibitors, or neutralizing antibodies. We sought to develop a novel, localized model of prolonged Wnt/ß-catenin signaling blockade by the application and validation of a lentivirus encoding ß-catenin short hairpin RNA (shRNA). Efficacy of lentiviral-encoded ß-catenin shRNA was first confirmed in vitro using bone marrow mesenchymal stromal cells, and in vivo using an intramedullary long bone injection model in NOD SCID mice. Next, the effects of ß-catenin knockdown were assessed in a calvarial bone defect model, in which the frontal bone demonstrates enhanced bone healing associated with heightened Wnt/ß-catenin signaling. Lentivirus encoding either ß-catenin shRNA or random sequence shRNA with enhanced green fluorescent protein (control) was injected overlying the calvaria of NOD SCID mice and bone defects were created in either the frontal or parietal bones. Among mice treated with lentivirus encoding ß-catenin shRNA, frontal bone defect healing was significantly reduced by all radiographic and histologic metrics. In contrast, parietal bone healing was minimally impacted by ß-catenin shRNA. In aggregate, our data document the application and validation of a lentivirus encoding ß-catenin shRNA model that represents an easily replicable tool for examining the importance of locoregional Wnt/ß-catenin signaling in bone biology and regeneration.