Category: Basic Sciences/Biologics; Ankle Introduction/Purpose: One of the most common procedures for treating end-stage osteoarthritis of the foot and ankle is arthrodesis 1. However, a significant complication with arthrodesis is the development of non-union. Thus, identification of adjuvants to enhance osteogenesis are paramount to fusion success. One possible molecule is the immunosuppressant FK506 (Tacrolimus). Previous research 2 demonstrated FK506 as a stand-alone small molecule capable of initiating osteogenesis and bone formation. The purpose of this study was to evaluate the osteogenic potential of FK506 alone and in combination with other bioactive factors to provide a foundation for future in vivo and clinical applications. Methods: Marrow-derived cells (MDCs) were isolated from juvenile bovine femoral condyles, to represent cells present at the ankle fusion site. MDCs (P1) were seeded at 50k cells per well (24-well plate). After 14 days, the wells were stained for calcium deposits using Alizarin Red S, and imaged with brightfield microscopy to visualize calcium deposits. Drug combinations of potentially osteogenic small molecules were tested (simvastatin, platelet derived growth factor PDGF, tamoxifen, triiodothyronine). The highest alizarin red absorbance values were used to choose the best drug combination. These combinations were further evaluated for gene expression by RT-qPCR Osteocalcin (OCN), Osteopontin (OPN), and Bone Sialoprotein (IBSP). To simulate a 3D osteogenic environment, cells were seeded into fibrin gels (to mimic the “fusion clot”), cultured for 14 days, and sectioned/stained with Alizarin red. Results: FK506 and PDGF produced the highest level of calcium staining compared to other bioactive factors, highlighted via heatmap of alizarin red absorbance values (Figure 1A). Compared to the control, FK506, PDGF, and the combination of both all resulted in enhanced alizarin red staining (Figure 1A/B). Overall, FK506 exhibited an increased expression across all genes. PDGF and the combination of FK506 and PDGF had a more varied expression within the genes queried (Figure 1C). Finally, in a fibrin gel system, variability was observed between gels of the same group. Displaying the best, middle, and worst sections give a comprehensive view of the explained variability. Across the sections, the FK506-PDGF combination showed the densest mineralization, highlighted by the dark maroon regions of the section (Figure 1D). Conclusion: This study confirms that FK506 demonstrates osteogenesis within marrow-derived cells (representative of a fusion site). The combination of FK506 to PDGF may enhance PDGF’s known healing potential within arthrodesis applications. This work provides the foundation for future clinical applications of FK506. Ongoing studies are focused on the effect of FK506, and potentially in combination with PDGF, in an rabbit ankle fusion model to evaluate efficacy and bone formation. The authors thank the American Orthopaedic Foot and Ankle Society (AOFAS) and Emory Department of Orthopaedics for their support and funding. References: 1 Park 2022. 2 Sangadala 2019