Effects of pore interconnectivity on bone regeneration in carbonate apatite blocks

Abstract

Porous architecture in bone substitutes, notably the interconnectivity of pores, is a critical factor for bone ingrowth. However, controlling the pore interconnectivity while maintaining the microarchitecture has not yet been achieved using conventional methods, such as sintering. Herein, we fabricated a porous block using the crystal growth of calcium sulfate dihydrate, and controlled the pore interconnectivity by limiting the region of crystal growth. The calcium sulfate dihydrate blocks were transformed to bone apatite, carbonate apatite (CO₃Ap) through dissolution-precipitation reactions. Thus, CO₃Ap blocks with 15% and 30% interconnected pore volumes were obtained while maintaining the microarchitecture: they were designated as CO₃Ap-15 and CO₃Ap-30, respectively. At 4 weeks after implantation in a rabbit femur defect, new bone formed throughout CO₃Ap-30, whereas little bone was formed in the center region of CO₃Ap-15. At 12 weeks after implantation, a large portion of CO₃Ap-30 was replaced with new bone and the boundary with the host bone became blurred. In contrast, CO₃Ap-15 remained in the defect and the boundary with the host bone was still clear. Thus, the interconnected pores promote bone ingrowth, followed by replacement of the material with new bone. These findings provide a useful guide for designing bone substitutes for rapid bone regeneration.

Keywords: bone regeneration; bone substitutes; carbonate apatite; pore interconnectivity.

Figure 1.

(A) XRD patterns and (B) FTIR spectra of CO₃Ap-30, CO₃Ap-15 and standard HAp. The miller indices of HAp were assigned from PDF# 00-009-0432.

Figure 2.

Photograph images of (A) CO₃Ap-30 and (B) CO₃Ap-15. μ-CT scanning observations of materials. μ-CT-based 3D reconstruction images of (C) CO₃Ap-30 and (D) CO₃Ap-15. μ-CT-based sliced images at a sagittal section of (E) CO₃Ap-30 and (F) CO₃Ap-15. μ-CT-based sliced images at a transverse section of (G) CO₃Ap-30 and (H) CO₃Ap-15. SEM images of (I) CO₃Ap-30 and (J) CO₃Ap-15. Magnified SEM images of (K) CO₃Ap-30 and (L) CO₃Ap-15.

Figure 3.

(A) Cumulative and (B) differential pore volume plotted as a function of pore diameter of CO₃Ap-30 and CO₃Ap-15. Differential pattern was best-fitted by two Lorentzian functions.

Figure 4.

Photograph images of materials after dye penetration assay. Perspective views of (A) CO₃Ap-30 and (B) CO₃Ap-15. Cross-sectional views of (C) CO₃Ap-30 and (D) CO₃Ap-15.

Figure 5.

(A) Compressive strength, (B) diametral tensile strength, (C) total porosity and (D) apparent porosity of CO₃Ap-30 and CO₃Ap-15. *P < 0.05.

Figure 6.

The amount of (A) calcium and (B) phosphorus released from CO₃Ap-30 (black circles) and CO₃Ap-15 (gray diamonds) to the HEPES buffer during 28 days of immersion measured using ICP-OES. *P < 0.05.

Figure 7.

Cell invasion assay of the samples with 3-mm thickness for 7 days of culture period. SEM images at the cell-seeded on-top surface of (A) CO₃Ap-30 and (B) CO₃Ap-15. SEM images at a cross-section of (C) CO₃Ap-30 and (D) CO₃Ap-15. SEM images at the bottom surface of (E) CO₃Ap-30 and (F) CO₃Ap-15. F-actins (red) and nuclei (blue) of attached cells on the bottom surface of (G) CO₃Ap-30 and (H) CO₃Ap-15. Magnified fluorescence images of the bottom surface of (I) CO₃Ap-30 and (J) CO₃Ap-15. Yellow triangles indicate cells.

Figure 8.

μ-CT images of rabbit femurs with defects reconstructed by grafting CO₃Ap-30 at (A) 4 weeks and (C) 12 weeks after implantation, and CO₃Ap-15 at (B) 4 weeks and (D) 12 weeks after implantation

Figure 9.

HE-Stained histological images of rabbit femurs with defects reconstructed by grafting (A) CO₃Ap-30 and (B) CO₃Ap-15 at 4 weeks after implantation. (C) and (D) show magnified versions of the images presented in (A) and (B), respectively. (E) and (F) show higher magnification images of typical regions in (A) and (B), respectively. (G) and (H) show TRAP-stained images in the region near (E) and (F), respectively. Yellow triangles, red triangles, blue triangles, ‘OB’, ‘OC’ and ‘#’denote new bone, red blood cells, adipose tissues, osteoblasts, osteoclasts and material, respectively.

Figure 10.

HE-Stained histological images of rabbit femurs with defects reconstructed by grafting (A) CO₃Ap-30 and (B) CO₃Ap-15 at 12 weeks after implantation. (C) and (D) show magnified versions of the images presented in (A) and (B), respectively. (E) and (F) show higher magnification images of typical regions in (A) and (B), respectively. (G) and (H) show TRAP-stained images in the region near (E) and (F), respectively. Yellow triangles, red triangles, blue triangles, ‘OB’, ‘OC’ and ‘#’denote new bone, red blood cells, adipose tissues, osteoblasts, osteoclasts and material, respectively.

Figure 11.

Area percentages of (A) bone and (B) remaining material in each bone defect. **P < 0.01.