| Since various types of bone defects,caused by trauma,cancer,physiological degeneration and other factors,are seriously detrimental to human health,the repair of bone defects is of urgency and importance.Due to different types of bone defects,the property requirements of bone repair materials are also different.So,the scaffolds with specific property that can match the requirements of bone defect become one of the key factors in the success of bone repair.In this thesis,a novel bioactive material,β-dicalcium silicate,was developed and studied.The ceramic,self-setting and composite scaffolds were prepared,and a series of fundamental studies on their material and biological properties were carried out.（1）Based on the ceramic properties ofβ-dicalcium silicate,in order to sastisfy the mechanical strength that injured weight-bearing bones restoration requires,this part of the thesis studied about preparation of high-strengthβ-dicalcium silicate scaffolds by pore-forming method and template method.The ceramic scaffolds were prepared through the pore-forming method and template method respectively.For pore-forming method,with the content of carbon particles of 50%,the particle size of 300-500μm and the sintering temperature of 1250°C,a ceramic scaffold with porosity of64.25%was fabricated.Osteoblasts MG-63 can favorably adhere and proliferate on the scaffold,illustrating the good biocompatibility of the scaffold.Meanwhile,the template method is also used to prepare a scaffold,whose compressive strength is found28.13 MPa at maximum.The simulated body fluid immersion experiment showed that the scaffolds induced the formation of dense apatite deposits on the surface of the scaffolds after soaking for 14 days,illustrating excellent bioactivity.Electron microscopy images revealed the good adhesion morphology of MG-63 cells.After 7days culture,MG-63 proliferated and migrated to the inside of scaffolds.Further more,Alamar Blue assay indicated that the extracted solution of scaffold could effectively promote cell proliferation.The PCR assay was applied to study the gene transcription of MG-63 on the ceramic or combining mechanical stimulation by a self-designed bioreactor.It was found that theβ-dicalcium silicate scaffold can promote cell adhesion by up-regulating the integrinβ1,COLI and FN.Both ceramic scaffold and combining mechanical stimulation can promote cell proliferation by up-regulating FN and TGF-β1,promote osteoblastic calcification by up-regulating ALP.Finally,subcutaneous experiment in nude mice has shown that theβ-dicalcium silicate scaffold has excellent ectopic osteogenic ablility.（2）Based on the self-setting property ofβ-dicalcium silicate,this part of the thesis studied about preparation of self-setting scaffolds at low temperature so as to build the basis for the further study of preparing scaffolds with complex structures.At first,the Pechini method is properly improved andβ-dicalcium silicate is fabricated with grain size about 120nm,where concentration of the free calcium oxide is less than 0.36%.Thereafter,dicalcium phosphate（DCPA）is doped to improve the self-setting property ofβ-dicalcium silicate.It showed that the strength of the material increased to the5.29MPa at max as 20%DCPA is added intoβ-dicalcium silicate（named as C8D2）with maintain for 1 day.Compared to pureβ-dicalcium silicate（named as C2S）,C8D2hydrate more quickly,whose hydration products are consisted of C-S-H,Ca（OH）2,HA and foshagite.The self-setting scaffold is fabricated using particle dissolution method with pore-forming agent Na Cl.The scaffolds have favorable connected pore structure with porosity about 75%and pore size from 200 to 500μm.Having been soaked in simulation body fluid for 14 days,the scaffold s exhibited good bioactivity since the crystal layers of HA and monetite were induced on the surface of scaffold.The scaffold also showed satisfactory biocompatibility.Osteoblast cells adhered and proliferated well on the scaffold surface while the extract liquid of s caffold could significantly promote the proliferation of MG-63 cells.After 7 days culture,the proliferation rates in the two groups,which are C2S and C8D2 leaching solution in turn,increased 17.5folds and 18.8 folds,respectively.C8D2 scaffold promot es the cell proliferation through up-regulating transcription of BMP-2 and BMP-6 in osteoblast cell,while up-regulating OPG and restraining transcription of IL-6 to improve the osteogenic activity and up-regulating transcription of ALP to improve osteogenesis calcification.（3）Functionalized dense-porous biphasicβ-dicalcium silicate/small intestinal submucosa（SIS）composite scaffolds were prepared through the regulation of drying-freeze drying process so as to block the migration of endothelial cells and fibroblasts by dense layer and improve osteogenesis by porous layer.The porous layer composite scaffolds have excellent connected pore structure whose pore size is about 100μm and aperture ratio about highly 94%as the SIS concentra tion is 0.8%and cryogenic temperature-20°C.Theβ-dicalcium particles can improve the hydrophilicity of SIS.The dense layer of the scaffold is composed of crisscross pattern of collagen fiber and the intervals between collagen are filled with extracellu lar matrix,which as a whole showed distinguished barrier effect.The HE staning showed that no endothelial cells HUVEC or fibroblasts HTFT migration to the porous layer.Osteoblasts were able to adhere and proliferate well in the porous layer of composite scaffold,indicating that the scaffold has excellent compatibility with osteoblasts.During the process of osteoblast growth,the composite scaffold can upregulate the transcription levels of t he proliferation related genes TGF-β1 and FN,the osteogenic activity related genes BMP-2,BMP-4 and BMP-6,and the osteogenesis mature related genes RUNX2,OSX and ALP,indicating its excellent biological activity.Overall,in this thesis,we designed and fabricated three types ofβ-dicalcium silicate bone repair scaffolds.The bioceramic scaffold has relatively high strength,the self-setting scaffold could self-set at low temperature meanwhile the composite scaffold has functionalized double-layer structure,whose charateristics may provide specialized properties for different bone repair strategies.These scaffolds with suitable porosity and pore structure,excellent biocompatibility and bioactivity are good for bone regeneration for their promoting osteoblast adhesion,proliferation and osteogenesis through regulating related genes,illustrating their capacity to apply in clinical as bone repair scaffolds. |
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