Perivascular Stem Cells Induce Paracrine-mediated Osteogenesis via Upregulation of Secreted Wnt-related Proteins

With an increase in the number of annual craniomaxillofacial orthopaedic procedures being performed, and an understanding of the drawbacks of autologous bone grafting, the bone graft substitute market is burgeoning. Several bone graft alternatives, including, FDA-approved osteogenic molecule, Bone Morphogenetic Protein-2 have shown promise in promoting large volumes of de novo bone formation, however are ladened with several adverse effects, hampering their clinical application. Perivascular Stem Cells (PSC) are a prospectively purified mesenchymal stem cell population isolated from adipose tissue by fluorescent-activated cell sorting (FACS), and have previously been shown to induce robust bone formation in small and large animal models. Herein, by utilizing species-specific MHC immunohistochemistry, we demonstrated that PSC elicit their osteogenic effect primarily via a paracrine-mediate mechanism. Subsequently, after resolving that PSC mediate host mesenchymal stem cells, via the secretion of pro-osteogenic molecules, RNA sequencing was performed to compare PSC with their unpurified predecessor, stromal vascular fraction (SVF). It was observed that Wnt-related secretory molecules, WISP-1 and Wnt16 were upregulated in PSC by 72- and 21- fold, respectively, when compared to SVF. Gain-of-function assays for these two secretory molecules revealed a significant increase in osteogenic markers, whereby a significant decrease in osteogenic markers was observed when WISP-1 or Wnt16 were knocked-down in PSC. Wnt16 was observed to indeed be a mixed ligand and demonstrate activation of both the canonical and noncanonical Wnt pathways. In aggregate, these data demonstrate that PSC promote osteogenesis via a primarily paracrine-mediate secretion of pro-osteogenic molecules, of which WISP-1 and Wnt16 are highly upregulated and play a functional and necessary role in PSC osteogenesis.