Three-dimensional printed calcium phosphate scaffolds emulate bone microstructure to promote bone regrowth and repair

Abstract

The interconnected structures in a 3D scaffold allows the movement of cells and nutrients. Therefore, this study aimed to investigate the in-vivo bioactivity of 3D-printed β-tricalcium phosphate (β-TCP) and hydroxyapatite (HAP) scaffolds that replicate biological bone. This study included 24-week-old male New Zealand white rabbits. A cylindrical bone defect with a diameter of 4.5 mm and a depth of 8 mm was created in the lateral aspect of the distal femur. A 3D-printed scaffold was implanted in the right femur (experimental side), whereas the left femur was kept free of implantation (control side). Micro-CT analysis and histological observations of the bone defect site were conducted at 4, 8, and 12 weeks postoperatively to track the bone repair progress. No evidence of new bone tissue formation was found in the medullary cavity of the bone defect on the control side. In contrast, on the experimental side, the 3D scaffold demonstrated sufficient bioactivity, leading to the growth of new bone tissue. Over time, new bone tissue gradually extended from the periphery toward the center, a phenomenon evident in both micro-CT images and biopsy staining. In the current study, we observed that the cells involved in bone metabolism adhered, spread, and proliferated on our newly designed 3D-printed scaffold with a bone microstructure. Therefore, it is suggested that this scaffold has sufficient bioactivity to induce new bone formation and could be expected to be a more useful artificial bone than the existing version.

Fig. 1

Structure of the 3D-printed scaffold. The trabecular bone area was emulated as biological bone, and the cortical bone area was a dense body with bony pores

Fig. 2

The upper row shows the surgical sites of the rabbit femur defect model. P, proximal; D, distal. The lower row shows 3D CT images of scaffold implantation in the femoral condyle of the rabbit

Fig. 3

a Region of interest (ROI) was defined as 4.4 and 4 mm in a coronal view. Volume/tissue volume, trabecular bone thickness, trabecular number, and trabecular separation were measured within the ROI. b The red areas in the 3D CT image indicated new trabecular bone formation

Fig. 4

The bone volume/tissue volume (BV/TV), trabecular bone thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) at 4, 8, and 12 weeks. The upper row is β-TCP group, and the lower row is the HAP group

Fig. 5

a Histological sections of β-TCP, HAP, and control with hematoxylin and eosin (HE) staining at 12 weeks. The black structure represents the scaffold, and the pale purple area along the structure represents the new bone. No new bone was observed in the blank control group (dotted rectangle). *, new bone; CB cortical bone, TB trabecular bone; S, scaffold. b Histological sections with HE and toluidine blue staining at 4, 8, and 12 weeks after implantation on the experimental side. The black structure is a scaffold of β-TCP or HAP. The β-TCP scaffold is resorbed over time and replaced by new bone, whereas the HAP remains in almost its original form at 12 weeks. In both groups, new bone formation was observed in the interior of the scaffolds after 4 weeks. c Histological sections of β-TCP with toluidine blue staining at 8 weeks in the high-magnification field. CN, new calcified bone; Oc, Osteocyte; Ob, osteoblast; Od, osteoid; S, scaffold. d Histological sections of β-TCP with ALP (left) and TRAP (right) staining at 4 weeks. Red arrows indicate osteoblasts (left), and blue arrows indicate osteoclasts (right). S, scaffold; NB, new bone