12. Optimizing VD3 Timing and Dosing for Significantly Increasing Fat Graft Retention in a Swine Model

Abstract

METHODS:Mice with a lineage-specific deletion of DDR2 (Pdgfra-CreER +/-;Ddr2 fl/fl ) and CreER -/controls as well as a congenital deletion of DDR2 (Ddr2 slie/slie ) and littermate controls (LC) received a 30% total body surface area dorsal burn with concurrent unilateral Achilles' tenotomy (burn/tenotomy, BT). C57B6 mice received BT and were treated with DDR2 inhibitor imatinib for 16 days post-injury to assess the role of DDR2 inhibition on HO. HO volume and collagen alignment were assessed by micro-CT and second-harmonic generation of collagen fibers, respectively. Confocal microscopy was performed to visualize expression of pFAK in MLin cells in vivo. Ddr2 slie/ slie and LC MLin cells were plated onto 3D collagen matrices or 2D collagen-coated plates to determine the effect of DDR2 on focal adhesion (FA) orientation and TAZ activation in vitro, respectively. Tamoxifen (TMX)-induced Pdgfra-CreER +/-;Ddr2 fl/fl and CreER -/control MLin cells were subjected to traction force microscopy (TFM) to determine strain forces on collagen-coated gels in vitro. TMX-treated Pdgfra-CreER +/-;Ddr2 fl/fl and WT MLin cells were treated with TRULI, an inhibitor of LATS, which phosphorylates and inhibits YAP and TAZ nuclear translocation, to define the role of DDR2 deletion in YAP/TAZ signaling in vitro. RESULTS:Previous analyses from our group identified Ddr2 as highly expressed and activated specifically in MLin cells following injury (omitted). Inhibition of DDR2 with imatinib decreased HO (A). Pdgfra-CreER +/-;Ddr2 fl/fl mice also showed reduced HO (B). Ddr2 slie/slie showed greater disorganization in collagen compared to LC following BT (C). FA analysis of vinculin showed altered FA orientation in Ddr2 slie/slie MLin cells which may explain the altered collagen alignment seen in vivo (D). TFM demonstrated the ability of MLin cells to provide strain on the ECM, though strain energy was similar between both groups (E). We hypothesized that DDR2 alters HO formation through a FAK and YAP/TAZ dependent process. Ddr2 slie/slie mice showed reduced activated pFAK in vivo at the HO site (F) and reduced nuclear (transcriptionally active) TAZ in vitro (G). TRULI-treated MLin cells with a DDR2 deletion showed reduced nuclear TAZ localization compared to WT MLin cells (H).CONCLUSION: These findings suggest that MLin-cell DDR2 signaling following injury promotes HO due to altered FA orientation and signaling through a FAK/YAP/ TAZ pathway to activate osteogenic differentiation. Collagen alignment alternations may be due to changes in FA orientation. Having identified the role of MLin DDR2 on HO and ECM alignment following injury, these results suggest that DDR2 may be an effective target in preventing aberrant MLin cell osteogenesis following injury.