Experimental Research Of Bone Tissue-engineering Induced Composite Scaffold In Repairing Bone Defects

Bone defects is one of common diseases in clinical, autologous bone graft as the gold standard to repair bone defect. However, due to limitations of autologous bone-derived severely restricted its use in clinical. In recent years, with the the advancement and development of tissue engineering and materials science, is expected to be able to effectively solve the bottleneck problem in clinical bone defect repair. Electrospinng is one of the effective methods for prepareing the current tissue-engineering scaffold, its productions have nano fiber structure and a certain porosity, faciliate the exchange of nutrients. Icariin is traditional Chinese medicine, it has made clear of its induced function.For these reasons, this study use Icariin, hydroxyapatite(HA), collagen(COL) and polycaprolactone(PCL), by the electrostatic spinning technology, freeze drying and genipin crosslinking to fabricate load Icariin drug microspheres CS/COL/PCL/HA composite scaffolds and no-load drug microspheres COL/PCL/HA composite scaffolds.Then with a series of experiments to evaluate the effect of composite scaffold on bone defect repair, hoping to become a new bone independent intellectual property rights repair materials. Includes the following contents: 1. Preparation and characterization of the composite scaffolds : Using Col / PCL / HA material to build the outer of composite scaffold by electrospinning, and using collagen to build the core of composite scaffold by freezing dryer, core embedded into outer, then put them into genipin solution to cross-linked. Materials science experiments to detect composition, microstructure, permeability, water absorption and porosity of composite scaffold; biomechanical evaluation of material mechanical properties, degradation in vitro analysis of the degradability of materials, and in vitro slow-release technology to evaluate microspheres drug release effect. Analysis datas, prove that the composite scaffold conform to the requirements of the tissue engineering scaffold material. 2. The co-culture experiment of different composite scaffold with MSCs: isolated the rat bone marrow mesenchymal stem cells and passage culture. Cells with different scaffold materials co-cultured for 3 days, 7 days and 14 days, take samples and fixed them, then use scanning electron microscope, histological staining(H&E staining) and cellular fluorescence staining to IV observe the cell adhesion, growth and proliferation in the scaffold material. 3. The scaffold material implanted into the host and observe the effect on repairing bone defect: copy the tibial platform(0.9 cm in diameter,0.5 in depth) defect experimental animal model, implant different scaffold into defect site. X-ray and three-dimensional CT to observe the osteogenesis effect after 4 weeks, 12 weeks and 24 weeks; histological staining(H&E staining and Masson staining) and immunohistochemical staining analysis and evaluate the degradation of scaffold materials, new bone formation and the effect of bone defect repair.The results show that the outer of composite scaffolds have nanofiber structure, good physical properties and degradation, the core of composite scaffolds have a porous structure, meet the requirements of the characteristics of the tissue engineering scaffold; cross-linked outer of water absorption and permeability rate are significantly higher than that of the nocross-linked(P<0.05), and the breaking load, maximum load and elastic modulus of the outer cross-linked are highly significant higher than that of the nocross-linked(P<0.01); cells with scaffold co-culture experiments show that cells can adhesion, growth and proliferation into the materials and scaffold non-toxic, and also the number of cells on load drug microspheres composite scaffold significantly more than them on no-load drug microspheres composite scaffold; Animal experiments have shown two species scaffolds can promote damage zone new bone regeneration, the two groups histological staining showed load drug microspheres scaffolds effects on bone repair significantly better than non-load drug microspheres scaffolds, immunohistochemical staining showed that at 12 weeks the load drug microspheres scaffolds group alkaliphosphatase(ALP) expression levels were significantly higher than 4 weeks, 24 weeks and no-load drug microspheres scaffolds group, load drug microspheres scaffold group of type I collagen(COL I), osteocalcin(OCN), osteopontin(OPN) expression levels were significantly higher than the no-load drug microspheres composite group.Synthesis the experiment results show that the loading drug microspheres CS/Col/PCL/HA composite scaffolds and Col/PCL/HA composite scaffolds have good biocompatibility, porous and osteogenesis function, its function of tissue induce, can be induced bone repair materials of clinical value by the later improvement with independent intellectual property rights. In summary, the results indicate that the load drug microspheres CS / Col / PCL / HA composite scaffolds have porous, good biocompatible, tissue-induced and better effect on bone repair, through subsequent improvement and further evaluation,the composite scaffold is expected to become bone repair material with independent intellectual property rights.