ABSTRACTThere are over two million long bone defects treated in the United States annually, of which ~5% will not heal without significant surgical intervention. While autogenous grafting is the standard of care in simple defects, a customized scaffold for large defects in unlimited quantities is not available. Recently, a three‐dimensionally (3D)‐printed bioactive ceramic (3DPBC) scaffold has been successfully utilized in the of repair critical‐sized (CSD) long bone defects in vivo. In this study, 3DPBC scaffolds were augmented with dipyridamole (DIPY), an adenosine A2A receptor (A2AR) indirect agonist, because of its known effect to enhance bone formation. CSD full thickness segmental defects (~11 mm × full thickness) defects were created in the radial diaphysis in New Zealand white rabbits (n = 24). A customized 3DPBC scaffold composed of β‐tricalcium phosphate was placed into the defect site. Groups included scaffolds that were collagen‐coated (COLL), or immersed in 10, 100, or 1,000 μM DIPY solution. Animals were euthanized 8 weeks post‐operatively and the radii/ulna‐scaffold complex retrieved en bloc, for micro‐CT, histological, and mechanical analysis. Bone growth was assessed exclusively within scaffold pores and evaluated by microCT and advanced reconstruction software. Biomechanical properties were evaluated utilizing nanoindentation to assess the newly regenerated bone for elastic modulus (E) and hardness (H). MicroCT reconstructions illustrated bone in‐growth throughout the scaffold, with an increase in bone volume dependent on the DIPY dosage. The histological evaluation did not indicate any adverse immune response while revealing progressive remodeling of bone. These customized biologic 3DPBC scaffolds have the potential of repairing and regenerating bone. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2499–2507, 2019