4-Aminopyridine Promotes BMP2 Expression and Accelerates Tibial Fracture Healing in Mice

Background:Delayed bone healing is common in orthopaedic clinical care. Agents that alter cell function to enhance healing would change treatment paradigms. 4-aminopyridine (4-AP) is a U.S. Food and Drug Administration (FDA)-approved drug shown to improve walking in patients with chronic neurological disorders. We recently showed 4-AP's positive effects in the setting of nerve, wound, and even combined multi-tissue limb injury. Here, we directly investigated the effects of 4-AP on bone fracture healing, where differentiation of mesenchymal stem cells into osteoblasts is crucial.Methods:All animal experiments conformed to the protocols approved by the Institutional Animal Care and Use Committee at the University of Arizona and Pennsylvania State University. Ten-week-old C57BL/6J male mice (22 to 28 g), following midshaft tibial fracture, were assigned to 4-AP (1.6 mg/kg/day, intraperitoneal [IP]) and saline solution (0.1 mL/mouse/day, IP) treatment groups. Tibiae were harvested on day 21 for micro-computed tomography (CT), 3-point bending tests, and histomorphological analyses. 4-AP's effect on human bone marrow mesenchymal stem cell (hBMSC) and human osteoblast (hOB) cell viability, migration, and proliferation; collagen deposition; matrix mineralization; and bone-forming gene/protein expression analyses was assessed.Results:4-AP significantly upregulated BMP2 gene and protein expression and gene expression of RUNX2, OSX, BSP, OCN, and OPN in hBMSCs and hOBs. 4-AP significantly enhanced osteoblast migration and proliferation, collagen deposition, and matrix mineralization. Radiographic and micro-CT imaging confirmed 4-AP's benefit versus saline solution treatment in mouse tibial fracture healing (bone mineral density, 687.12 versus 488.29 mg hydroxyapatite/cm³ [p ≤ 0.0021]; bone volume/tissue volume, 0.87 versus 0.72 [p ≤ 0.05]; trabecular number, 7.50 versus 5.78/mm [p ≤ 0.05]; and trabecular thickness, 0.08 versus 0.06 mm [p ≤ 0.05]). Three-point bending tests demonstrated 4-AP's improvement of tibial fracture biomechanical properties versus saline solution (stiffness, 27.93 versus 14.30 N/mm; p ≤ 0.05). 4-AP also increased endogenous BMP2 expression and matrix components in healing callus.Conclusions:4-AP increased the healing rate, biomechanical properties, and endogenous BMP2 expression of tibiae following fracture.