| Objective: The advancement of bone tissue engineering offers a new approach to repairing the clinical bone defects. Among those crucials in this field is the scaffold material. In my studies, the transformed-hydroxyapatite is made from the cuttlebone through hydrothermal reactions and the feasibility of applying it as the scaffold material in bone tissue engineering is also observed.Methods: (l)The cuttlebones, harvested from cuttles, undergo the chemical reaction with (NH4)2HPO4 in high temperature and high pressure for some time. Powder X-ray diffraction analysis (XRD), Fourier transform infrared spectroscopy (FTIR) and X-ray spectroscopy (XRS) are employed to characterize the cuttlebones and the outputs from the reaction. At the same time, the effects of different temperatures, pressures and time-lengths on the reaction are also observed. (2) Scanning electron microscopy (SEM) is employed to observe the spacial structure of the outputs in order to explore whether they have the needed network structure fit for the tissue-engineered scaffold material. (3) By inductively coupled plasma atomic emission spectrometer (ICP-AES) , the contents of the metal elements in the cuttlebone are tested to assess the toxicity. The biocompatibility of the outputs of the reaction is also systematically investigated, which included haemolysis test, cell culture cytotoxicity assay and cell adhesion test. (4)The marrow stromal osteoblasts (MSOs) are cultured in vitro and combined with theoutputs of the hydro-thermal reaction to form the MSO/CBHA compound artificial bones (MCCABs). The MCCABs are implanted into nude mice subcutaneously to observe their osteogenesis effects in immune-free animals. (5)The MCCABs are also auto-transplanted into the cranial defects in order that the abilities to repair bone defects are observed.Results: (1)The results of the XRD, FTIR and XRS analyses show that the chemical component of the cuttlebone is CaCO3 and the crystal type is aragonite. Under the condition of high temperature and high pressure, cuttlebones, together with (NH4)2HPO4, undergo a hydro-thermal reaction, and thus transform into hydroxyapatite-that is, the cuttlebone-transformed hydroxyapatite(CBHA). (2)The rate of the chemical reaction depends on the temperature and the pressure. Under the condition of 180癈 and 0.99MPa, it takes 2 days for the cuttlebones to transform totally into CBHA; under the condition of 250 C and 3.92MPa, 3 hours. (3) Within different lengths of time, the cuttlebones transform into the gradient materials with different rate of aragonite/HA. At 180 C, in 0.99MPa, and after 12- hour reaction, the rate of HA is 24%; after 1-day, the rate is 44%; and after 2-day, the rate is 100%. At 250 C,in 3.92MPa, and after 1-hour reaction, the rate of CBHA is 20%; after 2-hour reaction, the rate is 78%; and after 3-hour, the rate is 100%. (4)The SEM observation shows that under the low magnification the CBHA materials have the interconnected micro-porous network structures. Under the high magnification, CBHAs appear to have many micro-spheres, which are composed by nano-spheres, nano-silvers, nano-tubes and nano-granules, thus construct a new self-assembled nano-material system. (5)The rabbit haemolysis rates of the CBHAs are 0.004 and 0.005. The MSOs grow well in vitro with culture medium added with the extract of the CBHAs and they also adhere and proliferatewell on the surface of the CBHAs. These results show that CBHAs have good biocompatibility. (6)In nude mice, the ectopic osteogenesis of MCCAB is obvious and the new bone formation increases in pace with time. (7)The MCCAB can repair autogenous calvaria defects effectively and the effect is much better than that of CBHA alone and blank group.Conclusion: (1) The chemical component of the cuttlebone is CaCO3 and the crystal type is aragonite. (2)Under the condition of high temperature and high pressure, cuttlebone can transform into hydroxyapatite (CBHA). (3)The speed of the hydrothermal reaction is temperature-dependent and pressure-dependent.
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