Preparation, Characterization And Cytocompatibility Of Nano-β-Tricalcium Phosphate Porous Scaffold

Part I Preparation and Characterization of Nano-β-Tricalcium Phosphate Porous ScaffoldObjective:The porous scaffold plays a key role in tissue engineering because of the capability in promoting cell attachment, proliferation and differentiation. The construction and characterization of scaffold are related to biological performances. β-Tricalcium phosphate (β-TCP) scaffold has been demonstrated with good bone conductivity and cytocompatibility. It has played an important role in reconstruction surgery and bone dental implants. Bone tissue engineering should take much advantage of β-TCP due to its good capability. However, porous β-TCP scaffold could not meet the requirements of bone tissue engineering for lacking of strength and interconnection. Improving the intensity and interconnection of porous β-TCP scaffold has become the focus in bone tissue engineering. Up to now, there are tremendously numerous of methods to prepare porous β-TCP scaffold, but what makes them difficult to be operated under ordinary laboratory circumstances is that lots of preparations are complex and demandingly. A nanotechnologically optimized method which will be viable under ordinary laboratory conditions is what this research aims to.Methods:Nano-β-TCP powder was synthesized by precipitation method with Ca(N03)2·4H2O and (NH4)2HPO4, then was mixed with the porogen(NH4Cl). The mixed powder was pressed in a mold to get the flan. The flan was sintered by the programmed temperature to harvest the porous scaffold. The phase composition was studied by X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR).The porosity and interconnection of the scaffolds were characterized by hydrostatic weighing and water uptake methods. The compressive strength was tested by universal testing machine. And the micro structures were observed by scanning electron microscope (SEM).Results:The results of XRD and FTIR indicated that the phase composition was closely similar to standard β-TCP. Hydrostatic weighing method showed that the porosity of the scaffolds was73.47±0.21Vol%(n=5). Water uptake result was37.56±0.28Wt%(n=5). The SEM observation indicated that the pores were relatively interconnected and homogeneously distributed. Compressive strength of the scaffolds was7.98±0.05MPa(n=5) tested by universal testing machine.Conclusions:Nano-β-tricalcium phosphate scaffold with homogeneous, controllable pore structure and stable characterization could be fabricated by this optimized method.Part Ⅱ Cytocompatibility of Nano-β-Tricalcium Phosphate Porous ScaffoldObjective:To obtain an appropriate seed cells is another important part of tissue engineering. Adipose derived stem cells (ADSCs) are detected as a kind of adult stem cells which are, confirmed by in vitro experiments in past decade, believed to be self-renewal and differentiation potential, and have generated great interest by their relative abundance and easy access. It is widely accepted that the ideal tissue engineering scaffold should be compatible with the ADSCs. Studies recently have shown that the mechanical stress is one of foundational factors to stimulate cell proliferation and differentiation. The stimulating effect of dynamics clearly should not be ignored. The cytocompatibility of the nano-β-tricalcium phosphate porous scaffold with ADSCs will be detected, and the horizontal rotary dynamic seeding method will be built then compared with the regular method.Methods:Rabbit ADSCs were harvested as the seed cells. Cytotoxicity of the nano-β-tricalcium phosphate porous scaffold was assayed by CCK-8method. Cells seeded on the scaffolds were observed by scanning electron microscope on the day1,4and7, and cellular biocompatibility then was assessed. Horizontal rotary dynamic seeding method was used in the research group while the cells were directly added to the scaffolds in the control group. Proliferation of the seeded cells in the two groups was assayed and then compared by CCK-8on day1,4and7.Results:According to CCK-8assay, the percentage of cells proliferation in different concentrations of scaffold extracts cultivated with ADSCs was no lower than100%, which indicated no cytotoxicity. Rabbit ADSCs could attach to and extend both on the surface (day1) and in the pores (day4) of scaffolds, the extracellular matrix was detected on day7. The research group showed higher OD (optical density) values on the day4and7than those in the control group.Conclusions:The nano-β-tricalcium phosphate porous scaffold had suitable cytocompatibility and no cytotoxicity existed. The dynamic seeding method did no harm on the cytocompatibility. It might promote the cells proliferation in the short term.