Modulation Of Calcium Phosphate Crystal Growth By Small Organic Molecules

Synthesis of inorganic materials with specific morphologies and architectures has been attracting great attention due to their unique physical-chemical properties and biological effects. Calcium phosphate, as the main inorganic component of human hard tissue, has been widely used in the field of bone tissue reconstruction owing to its good biocompatibility and bioactivity. Calcium phosphate physical-chemical properties (e.g. solubility, thermostability and mechanical performance) and biological effects (e.g. protein adsorption and cell behavior) may significantly influenced by crystal morphology, size and chemical composition. Especially, calcium phosphate with nano-to micrometer sizes, with high specific surface areas, can enhance protein adsorption and promote cell growth. Therefore, it is significantly important to regulate calcium phosphate morphologies and architectures at the nano/micro-scale.In this study, biomimetic synthesis method was used. Under hydrothermal condition, we innovatively used small organic molecules, with symmetric structure and different functional groups, as the morphologies modulation to implement the effective control toward calcium phosphate growth, and prepared calcium phosphate powder and coating with different nano/micro-structures. Meanwhile, analysis of calcium phosphate crystal growth process modulated by small organic molecules was carried out. Additionally, we investigated protein adsorption and cell behaviors on nano/micro-structured calcium phosphate particles and coating. On this basis, we further studied the effect of low doses copper on calcium phosphate growth for effectively promote the bone defect regeneration. The main content and results are summarized as following:Firstly, using urea as a precipitating agent, the reaction parameters of preparing complete crystal shape and high crystallinity HA crystal were achieved by studying the impact of hydrothermal conditions on the crystal growth, such as temperature, time and the urea content. High crystallinity HA whiskers (width:～1 μm, the aspect ratio:50～100) were prepared using urea as a precipitating agent (0.56 M) at 150℃ for 3 h. The HA whiskers formation mechanism assisted by urea molecule was analysed and elaborated.Moreover, using urea as a precipitating agent, the effects of small organic molecules with similar molecular structure but different functional groups (inositol hexaphosphate (IP6), inositol (IS) and 1,2,3,4,5,6-cyclohexanehexacarboxylicacid (H6L)) on calcium phosphate growth were carried out under hydrothermal condition. The results showed that when IP6 concentration was greater than 1.7%, hollow microspheres with nanostructure was obtained and the nanostructure from plates turned into wires, further turned into spheres as the IP6 concentration increased. When IS was added into solution, the whiskers products occurred soft aggregation and gathered into cluster. When H6L was added, HA whisker turned into flower aggregates, and further into hollow microspheres with different nanostructures. The formation process of hollow microspheres were characterized by trans-mission electron microscope (TEM), X-ray diffraction (XRD) and Fourier transform infrared spectroscope (FTIR) and results showed that hollow microspheres formation processes assisted by IP6 and H6L were similar which both using "amorphous nanosphere" as templates, self-assembling and formation HA crystals, then internal "amorphous nanosphere" disappeared and left HA hollow microspheres. Additionally, protein adsorption results demonstrated that HA with nano/micro-structures may have a selective adsorption and controlled release on proteins. Cell experiments showed that spherical HA was more benefit for cell proliferation and differentiation comparing with plate-like HA.Based on above, due to its importance in bone tissue growth, angiogenesis and antibacterial functions, copper ions was added into calcium phosphate synthesis process for fabrication new functional calcium phosphate materials. Copper ions was incorporated into HA crystals by replacing calcium ions proved by X-ray photoelectron spectroscopy (XPS) and HA lattice parameter variation. Copper-substitution accelerated calcium phosphate degradation and changed crystal growth habit, resulting flower-like HA microspheres generation. When Cu/(Cu+Ca) (molar ratio) of the reaction solution was greater than 0.05, the thermal stability of the HA product was decreased. When IP6 concentration was lower than 1%, copper ions and IP6 played a synergistic role in regulating calcium phosphate growth. Bacteria experiments showed that when copper ions added was higher than 5%, copper-substituted calcium phosphate had a good antibacterial effect on both Escherichia coli and Staphylococcus aureus. Cell experiments proved copper-substituted calcium phosphate had no significant cell toxicity or adverse effect on bone marrow-derived mesenchymal stem cells (BMSCs) growth.Then, calcium phosphate was deposited on scaffolds assisted by H6L molecules. Calcium phosphate crystals on the scaffolds changed from plates to wires, then to spheres as H6L concentration increased. HA scaffold with calcium phosphate crystal deposited had an obvious calcium ions release, and the release rate of spherical crystals was greater than that of wire crystals, and the plates release rate was lowest. Protein adsorption experiments showed that scaffolds with nano/micro-structured surface had better protein adsorption ability than naked scaffolds. Cell experiments indicated that the nano/micro-structures on the scaffolds had an effect on cell ALP activity. The cell ALP activity on the spherical crystals was highest, lower on the wire-like or plate-like crystals and lowest on the naked scaffolds.In conclusion, small organic molecules addition can effectively regulate calcium phosphate growth and synthesize calcium phosphate particles and coating with different nano/micro-structures. And, nano/micro-structured calcium phosphate has a selective protein adsorption and plays a key role in controlling protein release. Cell differentiation behavior can be effectively regulated by modulating calcium phosphate nano/micro-structures. Moreover, copper (trace metallic elements) addition can control crystals growth, guiding synthesis of new architectures and functional calcium phosphate materials. Progress in this work provides an important reference for fabrication calcium phosphate with nano/micro-structures, also explores new ideas in construction micro-environment for bone tissue regeneration.