1. After 10 hr (w/ serum) After 24 hr (C/C) After 24 hr (T/T) B -T +DEX +T +DEX -T -DEX +T -DEX Examination of the Effects of Thrombin on the Migration of Osteogenic Cells Results Conclusions -Thrombin stimulates the migration of both primary bone marrow derived cells in osteogenic culture conditions and of an osteogenic cell line. -This provides evidence that thrombin may be interacting with the osteogenic population of the primary cells. -The migration response is mediated via thrombin’s active site. -The activity of the thrombin in culture quickly decreases, which is likely cell mediated. -The ability of thrombin (under Dex + conditions) to increase the capacity for bone formation at a distant site does not correlate with an increased number of migrating cells. This suggests that thrombin may specifically interact with osteoprogenitor cells, although more work is required to confirm this. Future Work -Examine % of cells expressing the thrombin PAR-1 receptor. -Investigate if thrombin can also stimulate chemotaxis of bone marrow derived osteogenic cells. -Investigate the mechanism that mediates differences in the modified Boyden chamber/bone nodule assay.Acknowledgements Jeffrey M. Karp, Molly S. Shoichet and John E. DaviesIntroduction Thrombin (Factor IIa) is a 36.5 kD serine proteinase that is generated in the penultimate step of the blood coagulation cascade from the circulating plasma protein prothrombin (Factor II). Once formed, thrombin initiates fibrin polymerization by cleaving fibrinogen. Thrombin has been demonstrated to stimulate migration of a variety of cell types (Bar-Shavit et al. 1983, Dawes et al. 1993, Wang et al. 1997), however no studies exist that examine thrombin's role on bone marrow derived osteogenic cell migration. Delivering thrombin or thrombin peptides from biodegradable scaffolds may help enhance bone regeneration within such scaffolds by promoting osteogenic cell invasion to, and throughout the defect site. In this study, a 2-D cell migration scratch wound model was used to examine the effects of thrombin on rat bone marrow derived adherent cells under osteogenic culture conditions. An osteogenic cell line was used to provide evidence that the observed effects applied to the osteogenic population of the primary cells.Hypothesis Bone marrow derived osteogenic cells will be stimulated to migrate by the addition of thrombin and this effect will be mediated by a direct interaction with thrombin’s active site.Objectives To study the effect of thrombin on bone marrow derived osteogenic cells to determine: a.If thrombin stimulates chemokinesis b.If the migration response is mediated via thrombin’s active site c.If thrombin can affect the extent of bone formationMethods Proliferation: A BrdU cell proliferation assay was employed. Isolation of Migration:To isolate the effects on cell migration, confluent cell layers were incubated with 20  g of mitomyacin C (MMC) for 30 min, which completely inhibited cell proliferation. Inhibition of Thrombin’s Active Site: D-Phe-Pro-Arg- chloromethylketone (PPACK) was used at 200 [nmol]. Activity of Thrombin: Chromogenic substrate S-2238 was used. The change in absorbance over time was determined at 405 nm. Controls:Negative:1% Bovine Serum Albumin Positive:15% Fetal Calf Serum Cell Migration / Bone Nodule Assay: To determine if the cells that thrombin stimulated to migrate could produce bone nodules, a modified Boyden chamber assay was employed. Statistical Analysis: For multiple comparisons, analysis of variance (ANOVA) was performed with the Tukey’s test. For single comparisons, a paired student t-test was used. Comparisons were made at significance levels of 95%. * All error bars represent  95% confidence interval Figure 3. Figure 3. When Mitomycin C was used to inhibit cell proliferation and isolate the migration response, results were consistent with those displayed in Fig 2 (results for the primary cells are shown). Cells are grown to confluency Cell layer is scratched with a pipette tip Cell layer subjected to different conditions An average leading front of migration is determined Scratch Wound Model Primary Bone Marrow Derived Cells Cultured in Osteogenic Conditions Rat Bone Marrow Derived Osteogenic Cell Line (RSB4D-C8 ) % of Control Leading Front of Migration Figure 4. Samples of media (containing thrombin) were examined over 24 hours from different culture conditions with S- 2238. Thrombin activity decreased quickly in the presence of cells. Media from cell line cultures without thrombin did not interact with the thrombin substrate. Fibrin Structural Properties Thrombin Affects Cell Invasion Receptor Ligand Interactions Cell Migration Focus of this work Low Thrombin High Thrombin 199% 134% 0.51.0 2.0 5.0 + 0.51.0 2.0 5.0 + n=3-8 n=4-8 Figure 2. Figure 2. When thrombin was pre-incubated with PPACK and added to the cells in the scratch wound assay (T=1 PP=200), the response was inhibited for both the primary cells and the cell line. % of Control Leading Front of Migration T=1 T=1 PP=200 + PP=200 T=1 T=1 PP=200 PP=200 + n=7n=3 Figure 1. Figure 1. Thrombin stimulated advancement of both (left) the primary cells and (right) the osteogenic cell line. A concentration of 1 [U/ml] was chosen for subsequent experiments. Dead Cells (R 2 = 0.07) No Cells (R 2 = 0.03) Primary Cells (R 2 = 0.84) Cell Line (R 2 = 0.89) (n=3) % of Thrombin Activity Remaining Time [hours] Stuart Rae, Raisa Yakubovitch & Professor Jaro Sodek. This work was financially supported by an ORDCF awarded to JED and by a University of Toronto Doctoral Award and an OGS awarded to JMK. Figure 5. After 10 hours of adherence to the filter, cells on the upper surface were removed and either 1%BSA or 1%BSA containing thrombin was added for 24 hours. (A) More bone nodules were observed on the underside of the filters when a pulse of thrombin was added. However, (B) no statistically significant difference was found between the number of cells observed on the bottom of filters treated with (T/T) and without (C/C) thrombin. # of Cells that Migrated Through the Filter A P< 0.04 n=3-7 # Bone Nodules Institute of Biomaterials and Biomedical Engineering University of Toronto, CANADA ibbm e 800 700 600 500 400 300 200 100 T=1T=1 PP=200 FSM P < 0.04 # 484 n=6