Modeling the effect of platelet releasate on the migratory behavior of perivascular cells

Abstract

Event Abstract Back to Event Modeling the effect of platelet releasate on the migratory behavior of perivascular cells Niloufar Khosravi1, Vanessa C Mendes1 and John E Davies1, 2 1 University of Toronto, Faculty of Dentistry, Canada 2 University of Toronto, Institute of Biomaterials and Biomedical Engineering, Canada Topographically complex implants surfaces enhance osteoconduction[1], the recruitment and migration of osteogenic cells to the implant surface. The progenitors of osteogenic cells are of perivascular origin, and osteoconduction is a key determinant in osseointegration. We, and others, have assumed that platelets activated on an implant surface provide the initial stimulus for osteoconduction. Here, we hypothesized that it would be possible to model perivascular cell migration caused by platelet releasate, and thus address the mechanism by which a created density gradient of platelet products modulates osteoconduction. Methods: Two in vitro experiments were conducted. First, 50,000 Human Umbilical Cord Perivascular cells (HUCPVCs), a characterized population of mesenchymal cells, were harvested at passage 3, and added to Boyden chamber inserts (8μm pores). Platelet lysate PL; Serum-free Lonza medium (sfLM); 1:1 PL+sfLM; or 50 or 100 ng/ml of either PDGF-BB or TGFβ in sfLM was added to the bottom of the wells. Migrated cells were counted after 18 hours. Second, cells were seeded at the density of 1.5×106 cells/ml in a chemotaxis microdevice (ibidi, Germany). Then either a density gradient was formed over the cell observation channel by adding PL(+) and sfLM(-) to the feeder reservoirs (+/-), or no gradient controls (+/+) and (-/-). Cells migratory behavior was monitored by Time-lapse video microscopy for 24 hours. Cell tracking was performed in ImageJ, chemotaxis and motility parameters, including the Forward Migration Indices (FMIx, FMIy), average speed, and P-value of the Rayleigh test, were calculated. Statistical analysis was performed in SPSS. Results and Discussion: The Boyden chamber assay showed that PDGF-BB stimulated migration of the cells in a dose-dependent manner, while TGFβ inhibited migration. Interestingly PL still stimulated migration in a dose dependent manner, and to a greater degree than PDGF-BB alone, although it contains 3.5 times more TGFβ than PDGF-BB. In the microdevice assay, P-values for the Rayleigh test, demonstrated the inhomogeneous distribution of cells after 24 hrs in the PL gradient compared to both controls (Figure 1). Furthermore, motility and chemotaxis measurements showed that the FMIx(+/-)—direction parallel to the gradient—was at least 4 times higher than FMIy(+/-), and also both FMIx/y of (+/+) and (-/-) controls. Nevertheless, the average cell migration speed of 2.88um/min (+/-) was not significantly different from that of controls 2.57 and 2.73um/min for (+/+) and (-/-) respectively (Figure 2). Clinical extrapolation of these findings would suggest that more osteogenic cells are stimulated to migrate to an implant surface due to preferential directionality rather than accelerating their migratory rate. Conclusion: Cell tracking analysis, including the Rayleigh Test indicates that the cytokine gradient created by PL does not accelerate the speed of cell migration, but it does affect directionality. Lindsey Fiddes, Centre for Microfluidic Systems, University of Toronto