Novel porous gradient HA/ZrO2 engineering bone carrying gelatin/chitosan slow-release hydrogel loading BMP-2 and BMSCs repair vertebra defect in rhesus macaque

Abstract

Objective To examine the bone regeneration capability of a novel bone graft substitute (BGS) of porous gradient HA/ZrO2 carrying BMP-2 and BMSCs in gelatin/chitosan slow-release hydrogel (BBGC-h) to lumbar vertebral body defect in rhesus monkey. Methods The BBGC-h was evaluated by scanning electron microscope for its morphological characteristics. The BGS with or without BBGC-h was implanted into the lumbar vertebral body defects in rhesus monkeys. Twenty-four healthy rhesus monkeys (5-7 years, 5-8 kg) were provided by the Kunming Institute of Zoology, Chinese Academy of Sciences. They were randomly divided into groups A (Test, BGC_BBCG-h, n=10), B (Control, BGC, n=10) and C (Autologous, n=4), respectively. The anteroposterior and lateral radiographs of the lumbar spine were conducted under anesthesia at 24 h, 8 weeks, and 16 weeks postoperatively. Micro-CT and histomorphometry were performed on samples collected from 2 of 10 samples in each group A and B to observe the newly formed bone volume at 8 and 16 weeks postoperatively. At 16 weeks postoperatively, bone matrix composition markers, including typeⅠcollagen, osteocalcin, osteopontin, basic fibroblast growth factor, alkaline phosphatase and vascular endothelial growth factor, were analyzed for groups A and B by RT-PCR and Western Blot. Biomechanical testing was performed on harvested vertebral samples of 4 animals from each of the subgroups and 4 additional non-operated healthy animals. Results The BGS revealed a three-dimensional network structure with uniformly distributed chitosan micro-spheres on the surfaces of the material and the interior of the pores. Hydrogel encapsulated BMSCs were visible in the pores of the BGS. 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 the two time points after surgery via Micro-CT scanning and histomorphology. The results of biomechanical testing indicated that the vertebral body compression strength was the smallest in the group B compared with those of the other groups and the non-operated healthy group (P <0.05), while the energy to maximal load of group A and group C were higher than those of group B. There were significant higher protein expression of typeⅠcollagen, OCN, OPN, bFGF, ALP and VEGF in group A than in group B (P <0.05). Conclusion There may be a promising to use BGS_BBGC-h to repair vertebral bone defects. The novel bone graft substitute has similar effects compared with autologous bone. Key words: Gelatin; Chitosan; Hydrogel; Bone morphogenetic protein 2; Mesenchymal stem cells; Ceramics; Tissue engineering