Controlled Fabrication Of Biomimetic Nano-bone Materials Based On A Novel Strategy Of Bi-template Co-assembly

Many natural biomaterials with perfect structures and unique properties have been created by nature through a long evolution, such as hard abalone shell, tough spider silk, sharp mouse teeth and high mechanical natural bone etc. Nowadays, the vital issue is to focus on the formation mechanism and assembled structure of these natural biomaterials. With the discovery on the nature of biomineralization, that is organic molecules direct mineralized deposit of inorganic crystals. Therefore, it becomes a very hotpot thesis to synthesize biomimetic materials based on template self-assembled strategy. This novel template-directed route is able to fabricate the materials not only on the bionic component, but on the bionic structure. The outstanding properties of natural materials are highly related to hierarchical structure. Thus, it should greatly enhance the specific properties of biomimetic materials via the bionics of hierarchical structure.In this study, a novel bi-template co-assembly strategy is firstly proposesd to synthesize a new biomimetic bone-like material. This route is a vital breakthrough based on the current basis of single-template. It is well known that the natural bone is outcome of multi-template co-assembly. Therefore, the bi-template strategy may be highly close to simulate the formation nature of the natural bone in comparison with single-template method. However, the number of active center is increased with the increase of number of template. Nucleation and growth of crystal may be distorted by such many active centers.In this dissertation, we firstly selected collagen and silk fibroin as co-assembled initial template to come true the synthesized system of biomimetic nanobone substitute. The two proteins are selected due to their similar structure, compensative properties and common directed function. The structural characterization and component analysis of the template protein were investigated by CD and SDS-PAGE. The results indicated that collagen and silk fibroin as two fibrous protein molecules exhibited great cooperative effect. In addition, collagen and silk fibroin are able to form a more stable secondary structure of co-assembly bi-templateCollagen-silk fibroin/hydroxyapatite biomimetic nanocomposite was prepared via bi-template co-assembled strategy. As a control, collagen-hydroxyapatite and silk fibroin-hydroxyapatite were prepared by single-template directed method. The results of characterization indicated the inorganic crystal was nano-scale and needle-like hydroxyapatite, which was very similar to the natural bone on the morphology and properties. Furthermore, TEM and SAED results suggested that the inorganic crystal was an oriented growth along the c axis of fibrous template. We also proposed a possible growth model about crystal growth directed by fibrous bi-template.Currently, bone marrow mesenchymal stem cells are one of the most potential seeded cells in the bone tissue engineering. The biocompatibility of the new biomimetic nano-scale bone materials, which were prepared based on bi-template co-assembly strategy, were further evaluated by the bone marrow mesenchymal stem cells. The results of cell assay indicated that this new material had good biocompatibility and were capable of supporting the adhesion, proliferation and differentiation of bone marrow mesenchymal stem cells.In addition, the osteogenic differentiation of bone marrow mesenchymal stem cells on the bi-template-induced biomimetic materials was investigated on the qualitative analysis of the cell level and the quantitive analysis of the molecular level. The qualitative staining results indicated that both the alkaline phosphatase (ALP) and the calcium nodule as two specific markers in the early and later stage displaced the positive staining results. The results of RT-PCR further confirmed that some specific genes could express in the different staged on the process of osteogenic differentiation. Typeâ… collagen as a constitutive expression gene expressed highly and steadily at every stage of osteogenic differentiation. Alkaline phosphatase as a specific gene expressed highly in the early stage, but lowly in the later stage. Moreover, osteocalcin and osteonectin as a specific gene expressed lowly in the early stage, but highly in the later stage.Lastly, the animal assay was further investigated to estimated the biocompatibility of the novel bi-template-induced materials in vivo. The results suggested that there was good biocompatibility and also no obvious acute immunity reaction. H&E staining results confirmed that this cell-material composites exhibited good ability of new bone formation in vivo and also were a great bone repaired substitute.