| BACKGROUND:Bone defects caused by variety of reasons (trauma, tumor, infection, etc.) has been a major orthopedic problem. The main treatment of bone defects were autologous bone graft, allogeneic bone graft. They all have their own drawbacks:the shortcomings of autogenous bone graft were the limited of resources, the infection of donor part, deformity and function loss, etc; allograft may transmitted diseases, and caused host immune rejection reaction. The emergence of tissue-engineered has provided a broad way to the bone defects,and found a suitable material for bone defects was a hotspot in bone tissue engineering research in recent years. An ideal materials should have:good biological compatibilty and degradation; bone conductivity and induced; mechanical strength; Plasticity; appropriate aperture to ensure that new bone tissue can growth in and the supplying of nutrient; the chemical surface and micro-structure that support differentiation of bone cell growth.A single material often did not have so many features, and thus the composite of different materials was increasingly, Materials Science and Engineering of QingHua University, using bionics principles, made a kind of injectable Plasticity composite which have good bone conduction, and bone-induced availability injectable nano-hydroxyapatite/ chitosan (nano-Hydroxyapatite/chitosan,nHA/CS) composite,90% porosity, pore size 50～200μm. The material has good initial stability and strength, which fit for the parts of Weight-bearing, but also fit for the parts of other types of bone defect filling, with the material degradation, the new bone formated, completed the conversed and shaped of the bones.However, the biocompatibility of the composite is poorly understood,and the effect to repair bone defect is unknown.OBJECTIVE:1,Obtaining primary bone marrow strom cells by density gradient centrifugation, The nHA/CS composite was co-cultured with the passage 3 cells of BMECs in vitro, To evaluate the biocompatibility of material.2,Created bone defect model On the rabbit's femoral condyle, implanted injectable nHA/CS composite in bone defect, the material effect on bone defects repairing was observed.To study the repair of bone defect with injectable nHA/CS composite.TIME AND SETTING:The observation was performed at the Central Laboratory of Zhujiang Hospital, Southern Medical University from August,2008 to November,2009.METHODS:1,The Methods of Biological Compatibility Experiment:Obtain primary bone marrow stem cells from the 2-week-old New Zealand rabbits femur and tibia bone marrow by density gradient centrifugation which was widely recognized, The nHA/CS composite was co-cultured with the passage 3 cells of BMECs.The BMECs group was served as the control group.Qbservated the growth and proliferation of cells which inoculated on the materials by contrast microscope, electron microscopy, cells adhered on the of surface of materials were counted after 1,2,4 h, and calculate the adhesion rate,cell cycle was observed by flow cytometry,and calculate the proliferative index of inoculated cells.2,The Methods Bone Defected Repaired Experiment:24 New Zealand white rabbits weight 1.5-2.0kg were established a defects on femoral with diameter of 7mm, a depth of 10mm. And divided into 4-week group, 8-week group,12-week group randomly. Injectable nHA/CS composite materials was implanted in the side of the bone defect as an experimental group, injectable CS materials was implanted on the other side as the control group.4,8,12 weeks later, observation were made as follow:①The activities of animal after implantation and the healing status of bone defect;②X ray, CT scan observated the repairing of bone defect and bone defect healing was assessed in accordance with CT score;③The material degradation, new bone formation, the pathological changes of bone defect healing process, were observed on the junction of bone and materials;④The continuous process of material degradation and new bone formation was observed by SEM..RESULTS:1,The Result of Biological Compatibility:①Microscope:The BMSCs grown on edge of material, the number of cells was increasing with time, the growth was in good condition.②SEM:The BMSCs grown well on the surface of nHA/CS, a lot of pseudopod from the cell body were found.③Adhesion rate:The attachment rate in the nHA/CS group was lower than that of the control group at 1 hour (P< 0.05); however, there was no significant difference after 2 and 4 hours(P> 0.05). After seeding,④Flow cytometry:After seeding, the proliferation of cell had no changes (P> 005). Normal diploid cells could be found in both groups, which indicated that nHA/CS had no influence to cell cycle.2,nHA/CS materials for experimental bone defect repair:①General observation:the rabbits were activity after operation,there were not rabbits occured fractured during the observation period in both group.The nHA/CS group:The edge of normal bone and material was still clear 4 weeks later;And the boundary was blur 8 weeks later, the surface was smooth, there was a little impedance on defect; The bone defects are fully restored the 12 weeks later, the impedance was similar with nomoral bone.The CS group:The materials was closely with bone tissue 4 weeks later, there was fibrous grown into materials; The boundary between material and bone tissue was blur 8 weeks later, the Surface was not smooth, there was little impedance on defect;Bone defect was not healed completely, some cortical defects, fibrous tissue filled after 12 weeks.②X ray:The nHA/CS group:A ring of light belt existed between the normal bone tissue and material, condyle defects can be seen 4 weeks later; The light belt between normal bone tissue and material was vague, the light transmittance in the defects slightly less than the host bone tissue 8 weeks later, The supracondylar bone defect was healed,there is no obvious difference between the bone tissue and bone defect in light transmission 12 weeks later.The CS group:The bone defect on the femoral condyle was clearly, light belt was clearly between the material and the surrounding tissue 4 weeks later; The bone defect was reduction compared with the previous, light transmittance of defect area is slightly less than ambient host bone tissue8 weeks later, Bone defects was decreased compared with previous, but not completely healed 12 weeks later.③CT examination:The nHA/CS group:The line between normal bone tissue and material is clear 4 weeks later, outer cortical was discontinuous; The line was blurred 8 weeks later, outer cortical was continuous; Bone defect was healed 12weeks later.The CS group:The bone defect was visible outer cortical was discontinuous 4 weeks later;Bone defect was smaller compared with the previous 8 weeks later; The bone defects,did not complete healed, some cortical discontinuity 12 weeks later.CT Comparison prompted that there is differences between the experimental group and control group in effects of bone repairing (P<0.05);④HistologyThe nHA/CS group:The new bone formation at the junction of material and host bone 4 weeks later, a lot of new bone was formated;And there was lamellar bone tissue formatted 8 weeks later; Harvard's system formed, the original defect was filled with new lamellar bone tissue, bone tissue continuous 12 weeks later.The CS group:The fibrous tissue growth into material, and lymphocytes can be seen around the material4 weeks later;A few of new bone was formed at the edge of host bone, new trabecular bone formation after 8 weeks later; A few of lamellar bone tissue formation 12 weeks later, fibrous tissue filled the defect.⑤SEM:In the experimental group part of the material degraded while the new bone formatted 4 weeks later, the materials was replaced by new bone 12 weeks later.CONCLUSION:①The BMSCs can be grown, proliferatd, differentiated on the surface of nHA/CS which indicated that the material has good biocompatibility.②The nHA/CS composite has better capacity on bone defect repairing compared with pure chitosan, played a good role in bone defect repair. |
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