| In general, tissue engineering utilizes living cells and biomaterials, as well as suitable biochemical and physical factors to create tissue-like structure through engineering and materials methods, aiming to repair or regenerate damaged tissue or organ. For tissue engineering purpose, large numbers of cells with appropriate phenotypes are needed in engineering the many different living tissues, and microcarrier culture has become a newly arisen cell culture technology, facilitating not only the acquirement of large number of cells but also the investigation of cell behavior in vitro. However, most of the materials used to manufacture microcarriers were polymer materials. Therefore, it is of great theoretical and practical significance to explore new microcarrier for tissue engineering and regenerative medicine. Hydroxyapatite (HA) has been extensively investigated for biomedical applications because of its favorable characteristics, including good biocompatibility, osteoconduction and excellent adsorption properties. Furthermore, some researchers described HA as a potential material of cell microcarrier. Unfortunately, the application was limited due to its large theoretical density. In addition, HA has been used in cell culture in the forms of HA-polymer composite microspheres, but lack of research on HA-coated inorganic material. Hollow glass microsphere (HGM) has been extensively investigated in many fields because of their excellent physical and chemical properties. With different surface modifications, the applications of HGM have been extended, such as photocatalyst, microwave adsorption and electromagnetic wave shielding materials.In this research, HA-coated HGM were prepared using a biomimetic process after three different surface modifications. The technical parameters optimization and mechanism of surface modification, structure and property characterization of HGM surface were carried out. Based on previous work, the emphasis was put on the factors influencing the deposition of HA coating on HGM, such as simulated body fluid (SBF) concentration, immersion time, solid/liquid ratio and activation of HGM. The phase composition, morphology, specific surface areas, microstructure and surface element of them were characterized by X-ray diffractmeter (XRD), fourier transform infrared spectrum analyzer (FTIR), field emission scanning electron microscope (FE-SEM), surface area analyzer (SSA), high resolution transmission electron microscope (HRTEM), X-ray photoelectron spectrometer (XPS), and scanning probe microscope (SPM), respectively. The MC3T3osteoblastic cells were cultured by HA-coated HGM, the cell morphology, cell proliferation rate, alkaline phosphate (ALP) activity and total protein were detected.The results revealed that HGM with the main composition of SiO2and Al2O3, had regular spherical structure and smooth surface morphology. Because of the formation mechanism, HGM structure was composed of amorphous phases and crystalline. Besides, the surface of HGM had higher chemical activity after treated with three different surface treatments, due to the breakage of smooth and dense surface structure of HGM, which were compounded of Si-O-Si and Si-O-Al. After pretreatment, a dense and uniform HA coating was successfully deposited on HGM. By analyzing the structure and performance of HA coating, it could be determined that the proper pretreatment was treated with1mol/L NaOH for2h at36.5℃.The immersion time, SBF concentration, solid/liquid ratio and activation treatment played vital roles in the formation of HA coating on HGM. The results showed that the coating thickness increased with the immersion time in SBF. Meanwhile, the coating thickness increased rapidly with the immersion rate about0.14μm/day when immersed within15days, and then the increase became obviously slow and reached a platform after18days. This was distributed to, after immersion for15days, the thickness of HA coating on the HGM was about2μm, which impeded the releasing of Si-OH acting as the nucleation sites of HA in the process. It also could be observed that more microcracks appeared on HA layers with the increased immersion time. Furthermore, HA coating was not formed on the surface of HGM until the concentration increased to1.5SBF, and the effect of SBF concentration on the HA thickness was trivial. In addition, a uniform HA coating was formed on the surface of HGM when the liquid/solid ratio was increased to150:1. From above, it could be deduced that the optimum immersion parameters were1.5SBF, immersion time of15days, and the liquid/solid ratio of150:1.The HA coatings with a bilayer structure were adjacent curled flakes joint together by the flake-like crystals, and the inner coatings were dense and uniform HA. Besides, the obtained HA coatings with the thickness of2μm and pore size of100nm were poorly crystalline and carbonated, similar to biological apatite. This kind of nanotopography was suggested to improve cell response and enhance mass transfer in the aggregates.Obvious changes took place on the surface morphology and specific surface area with the increase of calcination temperature, and highest total pore volumes (0.0359ml/g) and uniform porous structure composed of nanoparticles appeared when the temperature reached600℃. Moreover, extra phase (tricalcium phosphate, TCP) appeared because carbonated HA decomposed at700℃. With the increase of the calcination temperature, the partial carbonate was firstly released from HA, but apatite-type structure remained unchanged. When the calcination temperature arrived at700℃, the flakelets of porous HA coatings began to partially decompose into TCP nanoparticles. HRTEM micro structure of HA coating showed that a mass of amorphous phase and flakelets existed in the coating. Further, the HA coating had high crystallinity, and the rod like particles in situ replaced the flakelets.The MC3T3osteoblastic cells were cultured on two kinds of HA-coated HGM, before calcined (HA-w) and calcined at600℃for30min (HA-600). The results indicated that the two kinds of HA-coated HGM improved the attachment, migration and differentiation of MC3T3osteoblastic cells after culture for different times. However, much of the coatings of HA-600peeled off in the culture process because the bonding strength decreased after calcination. Moreover, the cells on the HGM surface were relatively flatter and tended to attach HA coating. It was obviously that HGM had inhibitory effects for proliferation of MC3T3osteoblastic cells.On the surface of HA-coated HGM, the cells displayed lamellipodia and filopodia extensions. MTT assay demonstrated that there was significant difference between HA-w and control group at4days. Meanwhile, the ALP activity test showed that the cells cultured on HA-w was significant greater than on the control group after21and28days. Therefore, the preliminary study on preparation and MC3T3osteoblastic cells culture of HA-coated HGM is successful, and could have potential use as microcarrier for large-scale cell culture. |
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