The treatment of large segmental bone defects remains a challenge as infection, delayed union, and nonunion are common postoperative complications. A three-dimensional printed bioresorbable and physiologically load-sustaining graft substitute was developed to mimic native bone tissue for segmental bone repair. Fabricated from polylactic acid, this graft substitute is novel as it is readily customizable to accommodate the particular size and location of the segmental bone of the patient to be replaced. Inspired by the structure of the native bone tissue, the graft substitute exhibits a gradient in porosity and pore size in the radial direction and exhibit mechanical properties similar to those of the native bone tissue. The graft substitute can serve as a template for tissue constructs via seeding with stem cells. The biocompatibility of such templates was tested under in vitro conditions using a dynamic culture of human mesenchymal stem cells. The effects of the mechanical loading of cell-seeded templates under in vitro conditions were assessed via subjecting the tissue constructs to 28 days of daily mechanical stimulation. The frequency of loading was found to have a significant effect on the rate of mineralization, as the alkaline phosphatase activity and calcium deposition were determined to be particularly high at the typical walking frequency of 2 Hz, suggesting that mechanical stimulation plays a significant role in facilitating the healing process of bone defects. Utilization of such patient-specific and biocompatible graft substitutes, coupled with patient's bone marrow cells seeded and exposed to mechanical stimulation of 2 Hz have the potential of reducing significant volumes of cadaveric tissue required, improving long-term graft stability and incorporation, and alleviating financial burdens associated with delayed or failed fusions of long bone defects.