Mandible Bone Repair In Rabbits With N,O-Carboxymethyl Chitosan/Hydroxyapatite Composites

Objective: A preliminary study of bone regeneration was carried out in order to investigate the soluble carboxymethyl derivative chitosan's bioactivity. The novel composites were prepared by cross-linking of N,O-carboxymethyl chitosan (N,O-CMCS)and hydroxyapatite particles(HAp). The plastic composites were used in vivo for repairing experimental rabbits'mandible defections. Following observations and evaluations were carried out to make clear the composites, osteogenesis characterics, and tried to find out the mechanisms of optimizing composites for bone regeneration. The mainly purpose of this experiment was to give a preliminary experimental evidence and to prove the novel composites as potential bone substitutions or bone tissue engineering scaffolds. Methods: (l).Material preparation: N,O-CMCS and HAp were mixed with various ratios of 2:1, 1:1 and l:2(w/w), and mixed with sterile distilled water, then the N,O-CMCS/ HAp composites were prepared. After preparation, the physical and chemical properties of the composites were preliminary studied. (2). At first, 36 adult rabbits were divided into 4 time groups randomly(9/group), then all bilateral mandibles(18 sides) of each group animals were divided into 6 subgroups(3 sides/group): A: N,O-CMCS group (3 X 4); B : HAp group(3 X 4); C : N,0-CMCS/HAp(2:l w/w) group(3 X 4); D: N,O-CMCS/HAp(1: 1 w/w) group (3 X 4); E: N,O-CMCS/HAp(l :2 w/w) group (3 X 4); F: post-operative control group (3 X 4). The mandible segmental defects were made in the left and light mandibular body of all the rabbits with 1.2cm in length and 1 .Ocm in wild. Group A was implanted in pure N,O-CMCS; group B was implanted in pure HAp, groupC , D, E implanted in N,O-CMCS/HAp composites with the ratio of 2:1, 1:1, l:2(w/w) respectively. After 2, 4, 8 and 12 weeks post-operation, the experimental rabbits analyzed by radiographic, following, the animals of every group were killed according to different operation period and the implantations were taken off and studied by gross observation histological analysis and scanning electron microscope(SEM). Results: In post-operation control group, almost no new bones could be seen. In pure N,O-CMCS group(group A) there were a little of new bones formation with plenty fibrous tissues ingrowthed. In group B, the paste of pure HAp migrated and dispersed after implantion, so that there were only a little of new bone formations. In N,O-CMCS/HAp composite groups(groupC, D and E), the new bone formations were effectively and obviously. The composites with more HAp weremore suitable for new bone formation. In contrast, the group with more N,O-CMCS the new formed bones were deficient in strength and tough. We also could see that new little blood vessels growth in the new bones. Conclusions: The soluble N,O-CMCS was biocompatible and biodegradable when implanted in vivo. Cross-linking composites of N,O-CMCS and HAp were plastic and porous. The diameter of the pores of the composites ranged from 50um to 400um according to the various ratio of N,O-CMCS and HAp, and they were suitable for the growth of osteoblasts and new little blood vessels as well as reformation of new bone tissues. In this experiment, the composite with more HAp group(group E) was more bioactive and the new formed bones appeared strength , tough and elastic tenacity as well. On the other hand, the preparation and storage of N,O-CMCS and HAp were easily and cheap, so that the N,O-CMCS/HAp composites were prosperously and worth of application in the domain of bone regeneration. This study implicated that optimized N,O-CMCS/HAp composites were suitable for bone substitutes or bone tissue engineering scaffolds.