Osteogenic Small Molecule FK506 for Enhanced Bone Formation in a Rabbit Ankle Fusion Model

Abstract

Non-union remains a significant complication following ankle fusion procedures, underscoring the need for effective osteogenic adjuvants to improve boney healing and fusion outcomes. This study investigates the osteogenic potential of FK506 (Tacrolimus), both in vitro and in vivo, for enhanced ankle fusion. Known orthobiologics in the market, such as recombinant human platelet-derived growth factor-BB (rhPDGF-BB), have been utilized to promote bone healing and were included in this study to assess their comparative and combinatory effects with FK506. Bovine marrow-derived cells (MDCs) were cultured in 2D and 3D environments to assess temporal markers of osteogenesis: RUNX2 nuclear localization (early), Alizarin Red S staining (intermediate), and mineral deposition with micro-computed tomography (late). A rabbit ankle fusion model (t = 4 weeks) was utilized to evaluate FK506's early in vivo efficacy. In 2D cultures, FK506 resulted in an increase, but rhPDGF-BB and the combination of rhPDGF-BB and FK506 yielded the highest mineral deposition. However, in 3D fibrin gel cultures, FK506 alone demonstrated the greatest bone volume and tissue mineralization. In vivo rabbit ankle studies revealed that while ankle joint fusion was not achieved, FK506-treated animals consistently showed substantial bone formation anterior to the joint, indicating its potential for localized osteogenesis in the ankle environment. This study demonstrates FK506's osteogenic potential as a standalone or combinatorial therapy. These findings highlight the need for further optimization of FK506 delivery methods and fixation strategies to maximize its clinical utility in ankle fusion procedures. Statement of Clinical Significance: The investigation of FK506 represents a potential pharmacological strategy for enhancing fusion healing and improving patient outcomes.

Keywords: Tacrolimus; ankle fusion; non‐union; osteogenesis.

Figure 1

(A) Quantification of Alizarin Red S Absorbance (t = 14d). (B) Staining and (C) Nuclear intensity quantification (a.u.) of Runx2. n > 38 per group. (t = 3d).

Figure 2

(A) Heatmap of combinatorial Alizarin Red S Absorbance (t = 14d). (B) Macroscopic photos of 2D Alizarin Red Stain (pre/post solubilization) and (C) quantification (t = 14d). n = 6 per group.

Figure 3

(A) Quantification of bone volume, radiopacity, and tissue mineral for 3D fibrin gels. n = 6 per group. (B) Macroscopic photos of Alizarin Red S histology, including red intensity heatmap.

Figure 4

(A) Screw inserted through medial malleolus into talus to fuse the rabbit ankle. (B) A total of 50 µL fibrin solution injected into the joint space and allowed to gel, without or with FK506. (C) Representative μCT and heatmap images and (D and E) quantification of bone volume and tissue mineral. n = 4–5 per group.

Figure 5

(A) Histological analysis (H&E, Safranin/O Fast Green, Goldner's Trichrome, Gomori's Trichrome) of control and (B) FK506 animals.