Effect of calcium carbonate nanoparticles, silver nanoparticles and advanced platelet-rich fibrin for enhancing bone healing in a rabbit model

Abstract

This study aimed to evaluate the efficacy of calcium carbonate nanoparticles (CCNPs) to induce new bone formation in a critical size segmental bone defect in rabbit's radius when used alone, combined with silver nanoparticles (AgNPs) as a paste, or as a composite containing CCNPs, AgNPs, and advanced platelet-rich fibrin (A-PRF). Thirty-six adult apparently healthy male New Zealand White rabbits aging from 5 to 6 months and weighting 3.5 ± 0.5 kg were used. The animals were divided into four groups; control group, CCNPs group, CCNPs/AgNPs paste group, and CCNPs/AgNPs/A-PRF composite group. The animals were investigated at 4, 8, and 12 weeks post-implantation in which the healing was evaluated using computed tomographic (CT) and histopathological evaluation. The results revealed that CCNPs/AgNPs paste and CCNPs/AgNPs/A-PRF composite has a superior effect regarding the amount and the quality of the newly formed bone compared to the control and the CCNPs alone. In conclusion, addition of AgNPs and/or A-PRF to CCNPs has reduced its resorption rate and improved its osteogenic and osteoinductive properties.

Figure 1

Computed tomography 3D images (Right) and cross sections images (left) in control (a), CCNPs (b), CCNPs/AgNPs (c) and CCNPs/AgNPs/A-PRF (d) at 4 weeks (1), 8 weeks (2) and 12 weeks (3) post implantation.

Figure 2

Showing (A) the normal density of the radius of the four experimental groups. It showed a marked increase with animal age with no significant difference among the experimental groups at each evaluation time-point (P = 0.766–0.937). (B) the AOI density of all examined groups at different time points (4,8, and 12 weeks). There is a significant difference between all groups (P = 0.000) throughout all time points (P = 0.000). At the end of the observation period the AOI density of group (D) (1386) was very close to the normal bone density (1444.3) followed by group (C) (1501). (C) Showing the BAD density of all examined groups at different time points (4,8, and 12 weeks). There is a significant difference between all groups (P = 0.001–0.004) throughout all time points (P = 0.000). As shown in the results group (D) had relatively least bone density and at the end of the observation period the BAD density (1434) was relatively the same as the normal bone density (1444.3).

Figure 3

The histopathological score showed great significant differences in the histopathological score at 4,8 and 12 weeks of the observation period among the four experimental groups (P = 0.000). At the end of the observation period (12 weeks), group B couldn't make complete healing although the presence of some newly formed bone, while group C&D showed complete healing with score = 6–7 respectively at the end of the observation period with a statistical difference (*) between them versus the two other groups (group B and control).

Figure 4

Microscopic appearance of the control group showed at 4 weeks post-operative (PO) (1) the defect filled with unorganized fibrous tissue (F) with a large number of chondroblasts and osteoblasts (black arrows) at the bony edges. At 8 weeks PO (2), the defect was filled with cartilaginous tissue formation (C) with mature chondrocytes within organized fibrous tissue (F) while at 12 weeks PO (3), the defect was filled with fibrocartilage (FC) characterized by mature chondrocytes (C) in separate lacunae within fibrous tissue bundles.

Figure 5

Microscopic appearance of CCNPs group (group B) showed at 4 weeks PO (1), the defect filled with the cartilaginous transition from fibrocartilage characterized by migration of a large number of chondrocytes (Co) in separate lacunae within the fibrous tissue bundles (F). At 8 weeks PO (2), the defect was filled with large hyaline cartilage (C) with few osteonal canals on the peripheries of the defect (O), while at 12 weeks PO (3), the defect showed an increase in osteonal canal (O) formation within the calcified cartilage with a high number of osteoblasts (OB) within the osteonal canals.

Figure 6

Microscopic appearance of CCNPs/AgNPs group (group C) showed at 4 weeks PO (1), the defect filled with cartilaginous transition phase with formation of hyaline cartilage (H) and multiple mature chondrocytes in lacunae. At 8 weeks PO (2), the defect showed formation of fibrous connective tissue and woven bone (W) migrating to replace hypertrophic chondrocytes (Co) at the cartilage bone junction, while at 12 weeks PO (3), the defect showed the formation of the haversian system from osteonal canals (O) and trabecular bone condensation into compact bone (B) with calcium deposition (Ca).

Figure 7

Microscopic appearance of CCNPs/AgNPs/A-PRF group (group D) showed at 4 weeks PO (1), the defect filled with multiple osteonal canals (O) formation with a large number of osteoblasts (OB) forming trabecular bone (T) with active calcium deposition. At 8 weeks PO (2), the defect showed multiple osteonal canals (O) with compact bone formation (B) and calcium precipitation (Ca), while at 12 weeks PO (3), the defect showed the formation of the haversian system (H) with ossification and dense compact bone formation (B) with calcium deposition (Ca).