Effects of chitosan scaffold along with royal jelly or bee venom in regeneration of critical sized radial bone defect in rat

Abstract

The aim of this study was to compare the efficacy of honey bee venom (BV) and royal jelly (RJ) alongside chitosan scaffold (CS) in improving radius bone defect in rats. A total of 60 full thickness radial bone defects with a length of 5 mm were created in 60 male Wistar rats. Six healthy radial bones (3 rats) were also assigned as normal control for biomechanical studies. The defects were left empty (untreated group) or were filled by the autograft (autograft group), CS (CS group), CS alongside the BV solution (CS-BV group), and CS alongside the RJ solution (CS-RJ group). Healing of the bone defects were evaluated clinically and radiologically on days 0, 28, 42 and 56 after operation while the biomechanical testing and histopathological examination were performed on the 56th day after surgery. The autograft was more radiopaque than the untreated and CS groups at the 28th, 42nd and 56th postoperative days (P<0.05). The CS-BV and CS-RJ groups showed significantly higher radiographic outcomes than the untreated and CS groups at the 56th post-operative day (P<0.05). The density of osseous tissue (DOT) and the osteocytes and osteoblasts count of the CS-RJ and CS-BV groups were significantly higher than the CS and autograft groups (P<0.05). The biomechanical results of the CS-RJ group were significantly superior to the autograft, while the biomechanical properties of CS-BV group were not significantly different with the autograft group (P>0.05). The scaffolds in CS group were observable in the surgical site after 56 days. There was no significant difference in radiographs, DOT, cartilage tissue and fibrous tissue, and also biomechanical performances of the CS-BV and CS-RJ groups at the 42nd and 56th day after surgery. The untreated and CS groups showed weakest biomechanical results among all groups. It could be concluded that both treatment strategies in the CS-BV and CS-RJ groups were appropriate and useful in treating critical bone defects.

Keywords: Bee venom; Bone healing; Chitosan; Royal jelly.

Fig. 1

Macroscopic and radiographic findings of the radial defects at 56 days after injury. In the gross evaluation, the defects in the autograft group were filled by hard connective tissue including cartilage and bone which connected to cut edges of the radius and the ulnar periosteum. In the CS-RJ and CS-BV groups the healing areas were filled with bone and cartilage tissues. While in the untreated defect group the defect area was mostly filled by loose soft connective tissues. The autograft group demonstrated significant superiority over the untreated defect group at the 56th post-operative day (P<0.05). The untreated defect and CS groups demonstrated significantly inferior bone formation in comparison to the autograft, CS-RJ and CS-BV groups at the 56th post-operative day (P<0.05). CS: Chitosan scaffold, CS-BV: Chitosan scaffold-Bee venom, and CS-RJ: Chitosan scaffold-Royal jelly

Fig. 2

Tissue sections from the radial bone defects at the 56th post-operative day. In the CS-RJ and CS-BV groups a remarkable superiority in bone formation and a significant decrease in cartilage and fibrous tissue is evident compared to the untreated, and CS groups. The scaffolds in the CS-RJ and CS-BV groups participated more effectively in healing than in the CS group. The scaffold was replaced by woven bone and hyaline cartilage in the CS-BV and CS-RJ groups, at the 56th post-operative day. Primary osteons were present very often in the newly formed osseous tissue which indicates early stages of remodeling in the CS-BV and CS-RJ groups. Cartilage and woven bone filled the distance between the radial bone edges in autograft group in which the graft is still visible in the defect area. The healing area, in the untreated group, is filled by loose areolar connective tissue, and minimum amounts of bone and cartilage tissue which are observed in the vicinity of the radial bone edges. Residual of the scaffolds still remain in the healing area of the CS group after 56 day of implantation while surrounded by fibrous connective tissue. Major part of the regenerated tissues consists of cartilage and osseous tissue, in the CS group (H&E staining). CS: Chitosan scaffold, CS-BV: Chitosan scaffold-Bee venom, and CS-RJ: Chitosan scaffold-Royal jelly, CT: Cartilage tissue, LCT: Loos connective tissue, FCT: Fibrous connective tissue, RBE: Radial bone edge, WB: Woven bone, BV: Blood vessel, CCT: Calcified cartilaginous tissue, HC: Hyaline cartilage, DCT: Dense connective tissue, BM: Bone marrow, R: Remnants of the scaffold, and PO: Primary osteon

Fig. 3

Density of fibrous, cartilage and osseous tissues in the regenerated tissue of different groups at 56 days post-injury. The autograft had the highest DCT and the defect demonstrated the highest DFT. The CS-RJ and CS-BV have the highest DOT (P<0.05). The least amount of DOT belonged to the defect group while the lowest DFT contributed to the CS-RJ group (P<0.05). CS: Chitosan, CS-BV: Chitosan-Bee venom, CS-RJ: Chitosan-Royal jelly, DCT: Density of cartilage tissue, DFT: Density of fibrous tissue, and DOT: Density of osseous tissue

Fig. 4

Biomechanical properties of the radius and ulnar bone complex, 56 days after injury. Maximum stress (a), Maximum load (b), bending stiffness (c), yield load (d), yield strain (e), and ultimate strain (f) included for all the untreated and treatment groups. CS: Chitosan, CS-BV: Chitosan-Bee venom, and CS-RJ: Chitosan-Royal jelly