713720 - Bioavailability of Exosomes: In Vitro and in Vivo Reciprocity?

Purpose: Exosomes are nanoscale vesicles that mediate cell-to-cell communication via exchanging molecular cargo between donor and target cells. Mesenchymal stem cell modification (MSCs) towards an osteogenic path can occur by uptake of exosomes from other cells. It is less clear whether vesicle placement in the absence of cells will facilitate site-specific delivery through acellular transfer of osteogenic activity. Additionally, it is not understood whether in vivo activity by standard laboratory assay will translate in an in vivo model. An electrospun fleece was combined with bone marrow-derived exosomes in the absence of cells to evaluate osteoinductive potential that might be thermo-stable, and be used in a biologically neutral collagen carrier. Comparisons were made of standard laboratory assay of osteoinductivity (OI), and in vivo expression in a mouse calvarial defect model.

Methods used: Electrospun Type-I collagen was prepared with and without hydroxyapatite (SpinPlant GmbH, Leipzig) as a foundation base for application of the bone marrow-derived exosomes. Individual discs of the collagen enhanced scaffolds (3-mm) were prepared and placed in a mouse calvarial skull defect. The animals were followed for 4 and 8 weeks to evaluate osteogenic integration, and adjacent tissue morphology. by ultrafiltration and subsequent differential ultracentrifugation (Beckman Optima XE-90 Ultracentrifuge equipped with a SW32 rotor) of the clarified supernatant. The purified exosomes were characterized by flow cytometry using coating latex beads with the nanoparticles and subsequently labeling these beads with known exosome markers, CD63, CD81 and CD9. Microscopic observation, quantitative assessment of OI with an alkaline phosphatase assay, and flow cytometry were used to evaluate the composition, the hybrid nature of the addition ot the nano-collagen fibers, and a cell assay was used to determine the osteoinductivity. Bridging of the critical size defect was the determinant of the in vivo success.

Summary of findings: Spine-derived exosomes positively expressed flow cytometry markers tested. ALP activity on the scaffold with HA demonstrated an approximate 10-fold increase to that of the collagen scaffold alone. While a dose-dependent effect, with higher doses of exosomes resulting in a greater amount of alkaline phosphatase expression, expression that exceeded that of the 50ng BMP-4 control. Dose escalation from 1.5, 2, and 4E9 resulted in similar increases in expression that was statistically greater with the combination of the fleece with the exosome component. Bone formation in the mouse calvarium did not demonstrate gap closure at 4 or at 8 weeks, but did demonstrate enhanced osteoclastivity and robust bone remodeling at the margins of the defect.

Conclusions: Bone marrow-derived exosomes dried into an electrospun fibrillar collagen demonstrated in vitro osteoinductive potential that might provide site-specific placement that could enhance biologic potential. With the capacity for ambient temperature storage, the provision of site-specific placement becomes a technical consideration. Placement of the human tissue derived exosomes in a transgenic mouse calvarial defect model did not demonstrate bridging bone across the defect. While the use of bone marrow as a surgical adjuvant is a daily occurrence in spine surgery, the translation across species was not fluent.