ObjectiveTo investigate the formation of nanostructure on cuttlefish bone transformed hydroxyapatite (CB-HA) porous ceramics and the effects of different nanostructures on the osteoblasts adhesion, proliferation, and alkaline phosphatase (ALP) expression.MethodsThe cuttlefish bone was shaped as plate with diameter of 10 mm and thickness of 2 mm, filled with water, and divided into 4 groups. The CB-HA in groups 1-4 were mixed with different phosphorous solutions and then placed in an oven at 120℃ for 24 hours. In addition, the samples in group 4 were further sintered at 1 200℃ for 3 hours to remove nanostructure as controls. The chemical composition of CB-HA were analyzed by X-ray diffraction spectroscopy, Fourier transform infrared spectrum, and inductively coupled plasma (ICP). The physical structure was analyzed using scanning electron microscopy, specific surface tester, and porosity tester. The MC3T3-E1 cells of 4th generation were co-cultured with 4 groups of CB-HA. After 1 day, the morphology of the cells was observed under scanning electron microscopy. After 1, 3, and 7 days, the cell proliferation was analyzed by MTT assay. After 7 and 14 days, the ALP expression was measured by pNPP method.ResultsX-ray diffraction spectrum showed that the four nanostructures of CB-HA were made of hydroxyapatite. The infrared absorption spectrum showed that the infrared absorption peak of CB-HA was consistent with hydroxyapatite. ICP showed that the ratio of calcium to phosphorus of all CB-HA was 1.68-1.76, which was consistent with hydroxyapatite. Scanning electron microscopy observation showed that the nanostructure on the surface of CB-HA in groups 1-3 were large, medium, and small cluster-like structures, respectively, and CB-HA in group 4 had no obvious nanostructure. There were significant differences in the specific surface areas between groups (P<0.05). There was no significant difference in the porosity between groups (P>0.05). Compared with group 4, groups 1-3 have more pores with pore size less than 50 nm. After co-cultured with osteoblasts, scanning electron microscopy observation and MTT assay showed that the cells in groups 2 and 3 adhered and proliferated better and had more ALP expression than that in groups 1 and 4 (P<0.05).ConclusionThe size of cluster-like nanostructure on the surface of CB-HA can be controlled by adjusting the concentration of ammonium ions in the phosphorous solution, and the introduction of small-sized cluster-like nanostructure on the surface of CB-HA can significantly improve the cell adhesion, proliferation, and ALP expression of the material which might be resulted from the enlarged surface area.
Objective To investigate the preparation and properties of the novel silica (SiO2)/hydroxyapatite (HAP) whiskers porous ceramics scaffold. Methods The HAP whiskers were modified by the SiO2 microspheres using the Stöber method. Three types of SiO2/HAP whiskers were fabricated under different factors (for the No.1 samples, the content of tetraethoxysilane, stirring time, calcination temperature, and soaking time were 10 mL, 12 hours, 560℃, and 0.5 hours, respectively; and in the No.2 samples, those were 15 mL, 24 hours, 650℃, and 2 hours, respectively; while those in the No.3 samples were 20 mL, 48 hours, 750℃, and 4 hours, respectively). The phase and morphology of the self-made HAP whisker and 3 types of SiO2/HAP whiskers were detected by the X-ray diffraction analysis and scanning electron microscopy. Taken the self-made HAP whisker and 3 types of SiO2/HAP whiskers as raw materials, various porous ceramic materials were prepared using the mechanical foaming method combined with extrusion molding method, and the low-temperature heat treatment. The pore structure of porous ceramics was observed by scanning electron microscopy. Its porosity and pore size distribution were measured. And further the axial compressive strength was measured, and the biodegradability was detected by simulated body fluid. Cell counting kit 8 method was used to conduct cytotoxicity experiments on the extract of porous ceramics. Results The SiO2 microspheres modified HAP whiskers and its porous ceramic materials were prepared successfully, respectively. In the SiO2/HAP whiskers, the amorphous SiO2 microspheres with a diameter of 200 nm, uniform distribution and good adhesion were attached to the surface of the whiskers, and the number of microspheres was controllable. The apparent porosity of the porous ceramic scaffold was about 78%, and its pore structure was composed of neatly arranged longitudinal through-holes and a large number of micro/nano through-holes. Compared with HAP whisker porous ceramic, the axial compressive strength of the SiO2/HAP whisker porous ceramics could reach 1.0 MPa, which increased the strength by nearly 4 times. Among them, the axial compressive strength of the No.2 SiO2/HAP whisker porous ceramic was the highest. The SiO2 microspheres attached to the surface of the whiskers could provide sites for the deposition of apatite. With the content of SiO2 microspheres increased, the deposition rate of apatite accelerated. The cytotoxicity level of the prepared porous ceramics ranged from 0 to 1, without cytotoxicity. Conclusion SiO2/HAP whisker porous ceramics have good biological activity, high porosity, three-dimensional complex pore structure, good axial compressive strength, and no cytotoxicity, which make it a promising scaffold material for bone tissue engineering.