Application of a novel gradient HA/ZrO2 engineering bone scaffold for repair of cervical fusion in rhesus macaque

Abstract

Objective To prepare the gradient HA/ZrO2 engineering bone scaffold and evaluate its bone repair capability in the cervical fusion of rhesus macaque. Methods Bone morphogenetic protein 2 (BMP-2) in gelatin/chitosan slow-release hydrogel was prepared by multiple emulsion cross-linking, and its biological features were examined. Mesenchymal stem cells (BMSCs) of rhesus macaque were isolated, cultured and identified. The novel gradient HA/ZrO2 engineering bone scaffold was prepared for carrying BMP-2 and BMSCs, and its morphological characteristics were observed. The cervical intervertebral disc of experimental segments in 24 healthy male rhesus macaques were replaced with the novel gradient HA/ZrO2 engineering bone scaffold carrying a gelatin/chitosan gel slow-release hydrogel loading BMP-2 and 3rd generation of BMSCs (group A, n=8), or the empty novel gradient HA/ZrO2 engineering bone scaffold (group B, n=8), or autologous iliac graft (group C, n=4) or nothing (group D, n=4). The anteroposterior and lateral radiographs of the cervical spine were taken at once, postoperative 8 and 16 weeks. Histomorphometry was performed on samples at postoperative 8 and 16 weeks, and biomechanical testing was performed on harvested vertebral samples 16 weeks after operation. Results Electron microscopy for gelatin/chitosan gel slow-release hydrogel loading BMP-2 revealed a three-dimensional network structure with uniformly distributed chitosan micro-spheres, and its encapsulation efficiency and drug loading rate of BMP-2 were decreased with time, 87.4%±0.9% and 58.2%±0.5% on day 1st respectively, 45.2%±0.6% and 30.1%±0.4% on day 15th respectively. The release rates of BMP-2 were 12.6%±0.11% and 55%±0.16% on day 1st and 15th respectively. BMSCs with diverse cell morphology were found under microscope. After osteogenic induction of the 3rd generation BMSCs, the results of alkaline phosphatase, von Kouza staining and its surface-specific antigen tests showed that it was consistent with the biological characteristics of BMSCs. According to radiography and histomorphological results, the newly formed bone volume in the interior of the pores in the group A was significantly higher than that in the group B at different time points after surgery. The results of biomechanical testing indicated that the maximal load, compression strength and energy to maximal load of the three groups were higher than those of normal group (all P values 0.05). Conclusions The novel gradient HA/ZrO2 engineering bone scaffold carrying a gelatin/chitosan gel slow-release hydrogel loading BMP-2 and 3rd generation of BMSCs can effectively promote cervical intervertebral fusion, and it has similar effects as the autologous bone in cervical fusion of rhesus macaque according to radiography, histomorphological manifestation and biomechanical testing. Key words: Tissue engineering; Cervical vertebrae; Biocompatible materials; Rhesus macaque; Mesenchymal stem cells; Osteoblasts; Bone morphogenetic protein 2; Bone regeneration; Zirconia-hydroxyapatites; Animal experiment