3D-printed Biomimetic Bioactive Glass Scaffolds for Bone Regeneration in Rat Calvarial Defects

Abstract

The pore geometry of scaffold intended for the use in the bone repair or replacement is one of the most important parameters in bone tissue engineering. It affects not only the mechanical properties of the scaffold but also the amount of bone regeneration after implantation. Scaffolds with five different architectures (cubic, spherical, x, gyroid, and diamond) at different porosities were fabricated with bioactive borate glass using the selective laser sintering (SLS) process. The compressive strength of scaffolds with porosities ranging from 60% to 30% varied from 1.7 to 15.5 MPa. The scaffold's compressive strength decreased significantly (up to 90%) after 1-week immersion in simulated body fluids. Degradation of scaffolds is dependent on porosity, in which the scaffold with the largest surface area has the largest reduction in strength. Scaffolds with traditional cubic architecture and biomimetic diamond architecture were implanted in 4.6 mm diameter full-thickness rat calvarial defects for 6 weeks to evaluate the bone regeneration with or without bone morphogenetic protein 2 (BMP-2). Histological analysis indicated no significant difference in bone formation in the defects treated with the two different architectures. However, the defects treated with the diamond architecture scaffolds had more fibrous tissue formation and thus have the potential for faster bone formation. Overall, the results indicated that borate glass scaffolds fabricated using the SLS process have the potential for bone repair and the addition of BMP-2 significantly improves bone regeneration.

Keywords: Bioactive borate glass; In vivo bone formation; Pore geometry; Porosity; Scaffold architecture; Selective laser sintering.

Figure 1

(A) Unit cells and scaffold architectures: Cubic, spherical, X, gyroid, and diamond, (B) representative optical images of 5 × 5 × 5 mm³ borate glass scaffolds with corresponding architectures at three different porosities used for compression tests, (C) representative scaffold of each architecture measuring 10 × 10 × 10 mm³ used to measure porosity, and (D) 10 × 10 × 10 mm³ diamond architecture scaffolds at four different porosities (34% – 61%).

Figure 2

(A) Unit cells and pore shapes of cubic and spherical architecture scaffolds, (B) pore volume variation of spherical and cubic scaffolds with porosity. Cylindrical extensions to the spherical pores were designed to maintain sufficient pore size for powder removal from the scaffold.

Figure 3

Compression test results: (A) Compressive strength of borate glass scaffolds with five architectures at different porosity levels. Vertical and horizontal error bars represent the standard deviations of measured compressive strength and porosities, respectively, (B) curve fitting of strength versus porosity fraction with R² value for all curves is >0.98.

Figure 4

(A) Compressive strength of soaked scaffolds after 1 week in simulated body fluids (SBF), (B) comparison of percentage strength reduction for scaffolds with different architectures at lower and higher porosities, (C) optical image showing the physical transformation of the scaffold surface after soaking in SBF for 1 week.

Figure 5

Scanning electron microscopy images of borate glass and silicate glass scaffolds at low and high magnifications after immersion in simulated body fluids for 1 week: (A and B) Borate glass outer surface morphology and rounded, irregular spheroid-like formations, (C and D) silicate glass scaffold surface and well defined needle-like crystalline structures at higher magnification.

Figure 6

Percentage reduction in compressive strength of a scaffold versus the ratio of total surface area to volume of different architectures.

Figure 7

Hematoxylin and eosin stained sections (left) and Masson’s trichrome stained sections (right) of calvarial defect regions with four different treatment groups: (A) Cubic scaffolds without bone morphogenetic protein 2 (BMP-2), (B) Cubic (left) and Diamond (right) scaffolds without BMP-2, (C and D) diamond scaffolds without BMP-2, (E and F) cubic scaffolds with BMP-2, and (G and H) diamond scaffolds with BMP-2. The arrows in the pictures point to the borders of the defect region. Dense color (pink in H&E and blue in trichrome) in sections indicates mineralized bone tissue, white/background color indicates remaining scaffold in the defect region. Red/maroon color in trichrome stained sections indicates new bone. There was no significant new bone tissue formation in defects treated with scaffolds without BMP-2. Defects treated with “scaffolds and BMP-2” show significant new bone formation. The difference in tissue formation between cubic and diamond scaffolds even with BMP-2 was not significant.

Figure 8

Percentage of new bone tissue formation in cubic and diamond scaffolds quantified based on the total defect area. The bone growth between scaffold designs with or without bone morphogenetic protein 2 (BMP-2) was not statistically different. The bone formation in defects treated with BMP-2 was statistically significant (P = 0).

Figure 9

Histological sections of defect regions treated without bone morphogenetic protein 2 after 6 weeks. (A) Hematoxylin and eosin (H&E) stained sections of diamond scaffold, (B) H&E stained sections of cubic scaffold with the inset figures showing fibrous tissue in the pores and arrows indicating osteoblast cells lining the edges of the diamond glass scaffold strut, (C and D) magnified images of different regions of diamond scaffold showing fibrous connective tissue, newly formed bone tissue, and remaining glass, (E) Masson’s trichrome stain showing pocket of mineralized bone tissue in the pore and the new bone tissue (red) surrounding the glass filament indicated by dotted arrow, (F) Trichrome stain showing mineralized bone tissue formed adjacent to host bone tissue and from the bottom side of the defect (above dura matter). N – new mineralized bone, O – original host bone, G – remaining glass, F – fibrous connective tissue.