| With the quickening pace of life, space expansion, and frequent traffic accidents, Bone tissue damage, fracture incidence increased year after year. Repairing Bone defects caused by variety of reasons (trauma, tumor, infection, etc.) is always exists in orthopaedic problem urgently solved. 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.Bone marrow stromal cells (BMSCs) as seed cells for bone tissue engineering is is the one studied most in a number of seed cells. BMSCs has many differentiation potential, in particular conditions it can differentiate to osteoblasts, cartilage and fat cells. Proliferative capacity, wide variety of sources, minimal wounded and little damage, that has attracted most attention.Studies show that about 20% of bone marrow mesenchymal stem cells are quiescent, (stay G0 phase), suggesting that bone marrow mesenchymal stem cells have a strong proliferative capacity. After 12 generations in vitro culture, BMSCs can still maintain a normal phenotype and telomerase activity in chromosomes, after 15 generations in culture still maintain their osteogenic differentiation potential.Bone tissue engineering, not only needs osteogenesis ability strong seed cells, but also needs more proper artificially extracellular matrix (namely cell carrier) materials.Due to the special physiological bone structure and mechanical property-es,bone tissue engineering materials as the extracellular matrix has special requirem-ents for materials. Ideal scaffold for bone tissue engineering should meet the follow-ing conditions:①have better biocompatibility, for cell adhesion, proliferation, non-toxic, less antigenicity.②Have effective active surface, provide a good microenvironment for cells to grow, proliferate, and secrete matrix; can activate cell the specific gene expression, maintain normal cellular phenotype expression.③have three-dimensional porous structure, the porosity of 90% or more is preferable, the internal surface, is conducive to cell adhesion and ingrowth, but also conducive to the discharge of nutrient metabolism.④have a certain degree of plasticity, easy to process into the required shape, and has a certain mechanical strength, so that the organization has formed the shape of the required.⑤have suitable biodegradable, the degradation rate should be adapt to the growth rate of tissue cells,the materials should be able to gradually degraded.Biological calcium phosphate ceramic-like materials (TCP), bioactive glass ceramic (BGC), biphasic calcium phosphate ceramics (HA/TCP) as the representative of biological materials, has good osteogenic potential. But there are defects such as brittle big degradation slow, affecting new tissue late rebuilt.Organic materials have many advantages such as good biocompatibility, biodegrad- able, absorbable, the absorption rate can be controlled, etc, but the poor hydrophilicity, weak cell adhesion, lack of cell surface recognition signal, and lack of biological cells interaction are also obvious. Polymer residual organic solvents can cause cell toxicity.Natural materials have good biocompatibility, with cell recognition signal, conducive to the proliferation of cell adhesion,but supplies are limited and sometimes difficult to reach; Artificial materials lack of cell signaling, however, it can mass production, design and control structure, mechanical properties and degradation time.Therefore, Single type of material is generally difficult to meet the requirement of bone tissue engineering scaffold materials, by means of a suitable method to several single material combination, complement each other to form a composite material.Injectable hydroxyapatite/chitosan (nano-Hydroxyapatite/chitosan, nHA/CS) composite material is a nano-materials with better conduction, and induction of bone, The greatest feature of nano-materials is high surface area and porosity, which have good bone conduction, and bone-induced availability,allowing adherence dependent cell adhesion on the material, growth and diffe- rentiation,The injectable nano-hydroxyapatite/chitosan (nano-Hydroxyapatite/chito- san,nHA/CS) composite, 90% porosity, pore size 50-300μm. 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. After the previous experimental study confirmed that the injectable Nha/CS has good biocompatibility。And bone marrow stromal cells(BMSCs) osteogenic differentiation has long been known.This study was intended to in vitro BMSCs, and coculture with the nHA/CS composite, to verify the biocompatibility of the composites,Making femoral condyle bone defect animal model in rabbit, To assess the ability of enrichment of mesenchymal stem cells and nHA/CS composite scaffold,to repaire bone defect femoral condyle bone defect in rabbits 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 the material.2,Experiment study of repairing bone defects with riched bone marrow derived mesenchymal stem cells compound with nHA/CS scaffold in rabbits:Created bone defect model of femoral condyle in rabbit's, implanted enriched BMSCs-nHA/CS composite in bone defect, the material effect on bone defects repairing was observed.To study the access the repairment ability of the composite.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 BMSCs.The BMSCs 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 2,4,8,12h, and calculate the adhesion rate,cell cycle was observed by CCK-8,and calculate the proliferative index of inoculated cells.2,Experiment study of repairing bone defects with richbone marrow derived mesenchymal stem cells compound with nHA/CS scaffold in rabbits:36 New Zealand white rabbits weight 1.5-2.Okg 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. Enriched BMSCs-nHA/CS composite materials was implanted in the side of the bone defect as an experimental group, injectable nHA/CS materials was implanted on the other side as the control group; four rabbits in each group did not implant any material on both sides as a 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 three-dimensional reconstruction;③Bone mineral density measured the experimental group and the control group.④The material degradation, new bone formation, the pathological changes of bone defect healing process, were observed on the junction of bone and materials.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 2hour (P<0.05); however, there was no significant difference after4,8and 12 hours (P> 0.05).④CCK-8:After seeding, the proliferation of cell had no changes (P>0.05). During the first 7 days Materials and control groups showed the same trend of gradual growth, the 7th reach to the maximum.The proliferation of cells did not change significantly.Normal diploid cells could be found in both groups, which indicated that nHA/CS had no influence to cell cycle.2\Experiment study of repairing bone defects with richbone marrow derived mesenchymal stem cells compound with nHA/CS scaffold in rabbits:①General observation and X ray:the rabbits were activity after operation,there were not rabbits occured fractured during the observation period in both group.The experiment 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 control 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.The blank group:4 weeks later there is no signs of healing; 8 weeks, fibrous tissue filling the defect cavity with no bone tissue filling; 12 weeks, there is hardening of the edge defect, defect cavity density was significantly below that of the surround-ing tissue, No obvious bony component repair.②CT examination:The experiment 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 control 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.The blank group:4 weeks later there is no signs of healing; 8 weeks, fibrous tissue filling the defect cavity with no bone tissue filling; 12 weeks, there is hardening of the edge defect, defect cavity density was significantly below that of the surrounding tissue, No obvious bony component repair.③Measurement of bone mineral density:4 weeks,8 weeks,12 weeks later,the differences between the experimental group and control group is significant in effects of bone repairing (P<0.05), At each time point the experimental group BMD more than the control group, the difference was statistically significant.④Histology:The experiment group:The new bone formation at the junction of material and host bone 4 weeks later, a lot of new bone was formed; 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 control group:. The fibrous tissue growth into material, and lymphocytes can be seen around the material 4 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, the defect was unrepaired.The blank group:After 8 weeks, there the fibrous callus, but the small amount of fibrous connective tissue covered with cavity; 12 weeks, there are the formation of granulation tissue, but no formation of osteoblasts.CONCLUSION:①The BMSCs can be grown, proliferatd, differentiated on the surface of nHA/CS which indicated that the material has good biocompatibility.②The enriched BMSCs-nHA/CS composite has better capacity on bone defect repairing compared with pure nHA/CS, played a good role in bone defect repair. while the pure nHA/CS composite has better capacity on bone defect repairing compared with blank group,which suggesting nHA/CS involved in bone repair materials. Enriched BMSCs-nHA/CS composite is effitive in repairing bone defects and may have a promising clinical application. |
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