Enhanced bone regeneration in rat calvarial defects implanted with surface-modified and BMP-loaded bioactive glass (13-93) scaffolds

Abstract

The repair of large bone defects, such as segmental defects in the long bones of the limbs, is a challenging clinical problem. Our recent work has shown the ability to create porous scaffolds of silicate 13-93 bioactive glass by robocasting which have compressive strengths comparable to human cortical bone. The objective of this study was to evaluate the capacity of those strong porous scaffolds with a grid-like microstructure (porosity=50%; filament width=330μm; pore width=300μm) to regenerate bone in a rat calvarial defect model. Six weeks post-implantation, the amount of new bone formed within the implants was evaluated using histomorphometric analysis. The amount of new bone formed in implants composed of the as-fabricated scaffolds was 32% of the available pore space (area). Pretreating the as-fabricated scaffolds in an aqueous phosphate solution for 1, 3 and 6days to convert a surface layer to hydroxyapatite prior to implantation enhanced new bone formation to 46%, 57% and 45%, respectively. New bone formation in scaffolds pretreated for 1, 3 and 6days and loaded with bone morphogenetic protein-2 (BMP-2) (1μg per defect) was 65%, 61% and 64%, respectively. The results show that converting a surface layer of the glass to hydroxyapatite or loading the surface-treated scaffolds with BMP-2 can significantly improve the capacity of 13-93 bioactive glass scaffolds to regenerate bone in an osseous defect. Based on their mechanical properties evaluated previously and their capacity to regenerate bone found in this study, these 13-93 bioactive glass scaffolds, pretreated or loaded with BMP-2, are promising in structural bone repair.

Fig. 1

(a) Optical image of 13–93 bioactive glass scaffold prepared by robocasting for implantation in rat calvarial defect. (b) Higher magnification SEM image of the scaffold showing the dense glass filaments and porous architecture in the plane of deposition (xy plane). Inset: SEM image in a plane perpendicular to the xy plane. The scaffolds had a porosity of 47 ± 1%, a pore width of 300 ±10 μm in the plane of deposition (xy plane) and 150 ± 10 μm in z direction.

Fig. 2

(a) SEM image of a bioactive glass scaffold after reaction for 6 days in 0.25 M K₂HPO₄ solution (60 °C; pH = 12.0). Inset: cross section of a bioactive glass filament showing the thickness of the converted surface layer. (b)–(d) Higher magnification SEM images of the surface of the converted layer formed by reaction for (b) 1 day, (c) 3 days, and (d) 6 days in the phosphate solution.

Fig. 3

X-ray diffraction patterns of the as-fabricated bioactive glass (13–93) scaffold, and the converted surface layer formed by reacting the bioactive glass for 1 day, 3 days, and 6 days in 0.25 M K₂HPO₄ solution (60°C; pH = 12.0). The diffraction peaks corresponding to a reference hydroxyapatite (JCPDS 09-0423) and the main cristobalite peak (JCPDS 39-1425) are indicated.

Fig. 4

(a) Amount of BMP-2 released a different time intervals from the as-fabricated scaffold (0d) and the scaffolds pretreated for 1day, 3 day and 6 days (1d; 3d; 6d; respectively) into a medium composed of FBS/PBS. Each scaffold was initially loaded with 1μg of BMP-2. (b) Cumulative amount of BMP-2 released from the scaffolds (as a fraction of the amount of BMP-2 initially loaded into the scaffolds) versus time.

Fig. 4

(a) Amount of BMP-2 released a different time intervals from the as-fabricated scaffold (0d) and the scaffolds pretreated for 1day, 3 day and 6 days (1d; 3d; 6d; respectively) into a medium composed of FBS/PBS. Each scaffold was initially loaded with 1μg of BMP-2. (b) Cumulative amount of BMP-2 released from the scaffolds (as a fraction of the amount of BMP-2 initially loaded into the scaffolds) versus time.

Fig. 5

Von Kossa stained sections (a1–d1) and H&E stained sections (a2–d3) of rat calvarial defects implanted for 6 weeks with bioactive scaffolds as fabricated (0d) and pretreated for 1 day, 3 days, and 6 days in aqueous phosphate solution (1d; 3d; 6d; respectively). (a3)–(d3) are higher magnification images of the boxed areas in (a2)–(d2). Scale bar =1 mm for (a1–d2) and 200 μm for (a3–d3). G = bioactive glass; NB = new bone; O = old bone; arrows indicate blood vessels, arrowheads indicate the edges of old bone.

Fig. 6

Von Kossa stained sections (a1–c1) and H&E stained sections (a2–c3) of rat calvarial defects implanted for 6 weeks with bioactive scaffolds pretreated for 1 day, 3 days, and 6 days in aqueous phosphate solution and loaded with BMP-2 (1 μg/defect) (denoted 1d + BMP; 3d+BMP; 6d+BMP, respectively). (a3)–(c3) are higher magnification images of the boxed areas in (a2)–(c2). Scale bar =1 mm for (a1–c2) and 200 μm for (a3–c3). G = bioactive glass; NB = new bone; O = old bone; M = bone marrow-like tissue, arrowheads indicate the edges of old bone.

Fig. 7

Percent new bone formed in rat calvarial defects implanted with scaffolds of 13-93 glass at 6 weeks postimplantation: as fabricated (0d); pretreated for 1 day, 3 days, and 6 days in aqueous phosphate solution (1d; 3d; 6d; respectively); pretreated 1 day, 3 days, and 6 days and loaded with BMP-2 (1 μg/defect) (1d+BMP; 3d+BMP; 6d+BMP, respectively). The new bone formed is shown as a percent of the available pore space in the scaffolds. (*significant difference compared to 0d; **significant difference compared to 0d, 1d, and 6d; p < 0.05).

Fig. 8

Percent bone marrow-like tissue (a) and fibrous tissue (b) formed in rat calvarial defects implanted with scaffolds of 13-93 glass at 6 weeks postimplantation: as fabricated (0d); pretreated for 1 day, 3 days, and 6 days in aqueous phosphate solution (1d; 3d; 6d; respectively); pretreated 1 day, 3 days, and 6 days and loaded with BMP-2 (1 μg/defect) (1d+BMP; 3d+BMP; 6d+BMP, respectively). (*significant difference compared to 0d; **significant difference compared to 0d, 1d, 3d and 6d; p < 0.05).

Fig. 8

Percent bone marrow-like tissue (a) and fibrous tissue (b) formed in rat calvarial defects implanted with scaffolds of 13-93 glass at 6 weeks postimplantation: as fabricated (0d); pretreated for 1 day, 3 days, and 6 days in aqueous phosphate solution (1d; 3d; 6d; respectively); pretreated 1 day, 3 days, and 6 days and loaded with BMP-2 (1 μg/defect) (1d+BMP; 3d+BMP; 6d+BMP, respectively). (*significant difference compared to 0d; **significant difference compared to 0d, 1d, 3d and 6d; p < 0.05).

Fig. 9

Back-scattered SEM images of rat calvarial defects implanted with bioactive glass scaffolds at 6 weeks postimplantation: (a), (b) as-fabricated scaffolds; (c), (d) scaffolds pretreated for 3 days in aqueous phosphate solution; (e), (f) scaffolds pretreated for 3 days in aqueous phosphate solution and loaded with BMP-2. (NB = new bone; G = bioactive glass). The approximate thickness of the HA surface layer on the pretreated scaffolds prior to implantation is shown in (d) and (f).