Abstract: BACKGROUND: Akebia saponin D can promote the proliferation and differentiation of osteoblasts, increase the activity and quantity of osteoblasts, and promote matrix calcification and callus growth. Akebia saponin D is mainly used to treat osteoporosis and promote fracture healing, and its application in bone defect repair is rare. 
OBJECTIVE: To observe bone defects repaired by loading the sustained-release microspheres containing Akebia saponin D onto the nano hydroxyapatite/chitosan scaffold. 
METHODS: The sustained-release microspheres containing Akebia saponin D were prepared by W/O/W method. The nano hydroxyapatite/chitosan scaffold of sustained-release microspheres containing Akebia saponin D (hereinafter referred to as the sustained-release scaffold) and the simple nano hydroxyapatite/chitosan scaffold (hereinafter referred to as blank scaffold) were prepared by freeze drying method to evaluate the in vitro drug release ability of sustained-release microspheres and the scaffold. Mouse-derived preosteoblast MC3T3-E1 was seeded on two kinds of scaffolds to analyze cell adhesion, proliferation, and differentiation. Individually cultured cells were used as controls. A 1.5-cm bone defect was made in the middle of bilateral radius of 24 adult New Zealand white rabbits. Blank scaffold and sustained-release scaffold were implanted, separately. Gross observation, Micro-CT imaging examination, and histological observation were performed at 4 and 12 weeks after operation.
RESULTS AND CONCLUSION: (1) Both the sustained-release microspheres and sustained-release scaffolds containing Akebia saponin D had sustained-release effects. The drug release rate of sustained-release scaffold was more stable and lasting. (2) CCK-8 assay demonstrated that the cell proliferation rate on the sustained-release scaffold was significantly faster than that on the blank scaffold and control groups (P < 0.05). Under the scanning electron microscope, mouse-derived preosteoblast MC3T3-E1 was covered on the scaffolds of the two groups, and the number of cells on the sustained-release scaffolds was more than that on the blank scaffolds. (3) At 7 and 14 days of culture, alkaline phosphatase activity and Runx2 mRNA expression were higher in the sustained-release scaffold group than those in the blank scaffold group (P < 0.05). At 21 days of culture, osteopontin and osteocalcin expression levels in the sustained-release scaffold group were higher than those in the blank scaffold group (P < 0.05). (4) The results of imaging and histological observation showed that at 4 weeks after operation, a large number of new bone were found around the materials in the sustained-release scaffold group, and the new bone mass was significantly higher than that of the blank scaffold group. At 12 weeks after operation, a large number of new bones grew in the sustained-release scaffold, while only a small amount of new bones was found in the blank scaffold, and the material residue in the sustained-release scaffold group was significantly lower than that of the blank scaffold group (P < 0.05). (5) These results verify that nano hydroxyapatite/chitosan scaffold of sustained-release microspheres containing Akebia saponin D can enhance the adhesion, proliferation, and differentiation of osteoblasts in vitro, and promote the osteoinductive ability of nano hydroxyapatite/chitosan scaffold in vivo.

Key words: Akebia saponin D, nano hydroxyapatite, chitosan, scaffold, bone biomaterials, bone defect repair, bone tissue engineering