Regional gene therapy with 3D printed scaffolds to heal critical sized bone defects in a rat model

Abstract

The objective of the present study was to assess the ability of transduced rat bone marrow cells (RBMCs) that overexpress BMP-2 loaded on a three-dimensionally (3D) printed scaffold to heal a critical sized rat femoral defect. Tricalcium phosphate (TCP) scaffolds were 3D printed to fit a critical sized rat femoral defect. The RBMCs were transduced with a lentiviral (LV) vector expressing BMP-2 or GFP. The rats were randomized into the following treatment groups: (1) RBMC/LV-BMP-2 + TCP, (2) RBMC/LV-GFP + TCP, (3) nontransduced RBMCs + TCP, (4) TCP scaffold alone. The animals were euthanized at 12 weeks and evaluated with plain radiographs, microcomputed tomography (micro-CT), histology, histomorphometry, and biomechanically. Each LV-BMP-2 + TCP treated specimen demonstrated complete healing of the femoral defect on plain radiographs and micro-CT. No femurs healed in the control groups. Micro-CT demonstrated that LV-BMP-2 + TCP treated femoral defects formed 197% more bone volume compared to control groups (p < 0.05). Histologic analysis demonstrated bone formation across the TCP scaffold, uniting the femoral defect on both ends in the LV-BMP-2 + TCP treated specimens. Biomechanical assessment demonstrated similar stiffness (p = 0.863), but lower total energy to failure, peak torque, and peak displacement (p < 0.001) of the femurs treated with LV-BMP-2 + TCP when compared to the contralateral control femur. Regional gene therapy induced overexpression of BMP-2 via transduced RBMCs combined with an osteoconductive 3D printed TCP scaffold can heal a critically sized femoral defect in an animal model. The combination of regional gene therapy and 3D printed osteoconductive scaffolds has significant clinical potential to enhance bone regeneration.

Keywords: 3D printing; bone; regional gene therapy; scaffold; tissue engineering.

FIGURE 1

(a) Stereolithography (STL) file created from a computed tomography (CT) scan of a rodent femur. (b) Tricalcium phosphate (TCP) scaffold with 700 μm pores 3D printed to fit a critical sized rat femoral defect. (c) Intraoperative photograph demonstrating placement of the 3D rinted TCP scaffold (yellow arrow) within the rat femoral defect (the blue arrow points to the distal femoral segment and the red arrow points to the proximal femoral segment)

FIGURE 2

Representative plain radiographic images from groups 1–4 taken at 12 weeks after insertion of the 3D printed TCP scaffold. Only group 1 demonstrated complete healing of the defect. The yellow arrows illustrate bone surrounding the scaffold, bridging the defect proximally and distally

FIGURE 3

Representative micro-CT scans with three-dimensional reconstructions (top) and axial images (bottom) obtained from groups 1–4 after specimen harvest. Only group 1 demonstrated complete healing of the defect and on the axial image; circumferential bone can be seen surrounding the 3D printed TCP scaffold. No bone formed within the defect in groups 2–4

FIGURE 4

Select longitudinal histological cuts of the proximal scaffold-defect interface from groups 1–4. The samples are stained with Masson’s trichrome stain. Only group 1 demonstrated trabecular bone formation (yellow arrows) within the 3D printed TCP scaffold (white arrows), uniting the scaffold to both ends of the defect. There is no notable trabecular bone in groups 2–4, and the TCP scaffold (white arrow) is mostly surrounded by muscle

FIGURE 5

Plain radiograph demonstrating scaffold breakdown (yellow arrow) and proximal pin loosening with osteolysis at the proximal aspect of the bone defect (white arrow)