A Mechanistic Study of the Phase Transformation of Calcium Phosphate and Its Implication in Biomineralization

We have assigned the 31P high-resolution spectrum of octacalcium phosphate by 31P double quantum and HETCOR spectroscopy. The finite pulse RFDR sequence was used effectively for 31P double quantum NMR spectroscopy at a spinning frequency of 10 kHz. The 31P NMR data measured for hydroxyapatite and octacalcium phosphate show that sizable double-quantum excitation efficiency can be obtained with the ratio of the recoupling field to spinning frequency set equal to 1.67. The 31P peaks at -0.2, 2.0, 3.3 and 3.7 ppm are assigned to P5/P6, P3, P2/P4 and P1 sites in OCP, respectively. Our data reveal that substantial amount of the PO4 3- groups at the P2 and P4 sites have been transformed to HPO42- in our octacalcium phosphate sample. (Chapter 3) Based on the chemical shift data of OCP, we were able to study the molecular mechanism of OCP to hydroxyapatite (HAp) transformation in vitro by several physical techniques, with particular emphasis on solid-state 31P homonuclear double-quantum (DQ) NMR spectroscopy. The in vitro system is prepared by mixing urea, sodium phosphate monobasic dehydrate, and calcium nitrate tetrahydrate at 100 °C. The images obtained by scanning electron microscopy and transmission electron microscopy show that the blade like OCP crystals will transform into hexagonal rod-shaped HAp crystals as the pH of the reaction mixture increases slowly from 4.35 to 6.69 in 12 h. Together with computer-assisted lattice matching, our DQ NMR data reveal that OCP crystals transform to HAp topotaxially, where the [ ]HAp and [ ]HAp axes are along the same directions as the [001]OCP and [010]OCP axes, respectively. On the basis of our in vitro results, the formation of the central dark line commonly found in biological hard tissues could be explained by the inherent lattice mismatch between OCP and HAp. (Chapter 4) In addition to synthetic crystals, the structure and composition of the biological calcified tissues (rat dentine) were also analyzed by several advanced solid-state NMR techniques without any chemical pretreatment. The measurements of OH- content of calcium phosphates in the rat dentine were made by solid-state NMR spectroscopy under magic-angle spinning. The 31P{1H} (HETCOR) and the Lee-Goldburg homonuclear decoupling technique were combined to provide an efficient suppression of 1H-1H spin diffusion during polarization transfer. The analyses were carried out and repeated for different contact times in order to extrapolate the amount of OH- for each sample. Dentine samples of rats at different ages were studied, viz. 3 weeks, 5 months and 24 months. The OH- content of the rat dentine was found to decrease as the teeth become mature. The teeth aging also results in different cp values for HPO42- site in the biological samples. To enhance the spectral resolution, we combine the HETCOR experiment and the DQ experiment to extract the 31P homonuclear second moment of 31P signals with different proton environments. Based on the DQ results, the density of phosphate groups in the dentine was found to increase as the rat dentine become mature. (Chapter 5)Contents Chapter 1 Introduction 1 1.1 The Concept of Biomineralization 1 1.2 The Principles of Biomineralization 3 1.2.1 Biologically Induced Mineralization 3 1.2.2 Biological Controlled Mineralization 4 1.3 Types and Functions of Biominerals 6 1.3.1 Calcium Carbonate 6 1.3.2 Calcium Phosphate 8 1.4 The Structure and Composition of Calcified Tissues11 1.4.1 Bone 11 1.4.2 Teeth 14 1.5 Solid-State NMR Studies of Calcium Phosphate Minerals16 1.6 Scope of this Thesis 17 1.7 References 18 Chapter 2 Basic Nuclear Magnetic Resonance Principles 21 2.1 Introduction 21 2.2 Quantum Mechanical Description of NMR 21 2.2.1 Nuclear spin 21 2.2.2 The Zeeman Interaction 23 2.2.3 The Density Matrix 24 2.2.4 The Density Matrix in Thermal Equilibrium 25 2.2.5 Equation of Motion of the Density Operator 27 2.3 A Simple NMR Experiment 28 2.4 Internal Interactions 31 2.5 Magic Angle Spinning 34 2.6 Interaction Frame 36 2.7 Cross Polarization 37 2.8 Lee-Goldburg Irradiation 40 2.9 Multiple Quantum Coherences 42 2.10 Homonuclear Radio Frequency-Driven Recoupling 43 2.11 References 45 Chapter 3 Solid State NMR study of Octacalcium Phosphate47 3.1 Introduction 47 3.2 Experimental Section 49 3.2.1 Materials 49 3.2.2 Synthesis of Octacalcium Phosphate (OCP) 49 3.2.3 Characterization 49 3.2.4 Numerical simulation 52 3.3 Results 54 3.3.1 XRD and FT-IR 54 3.3.2 SEM, TEM and Electron Diffraction (ED) 55 3.3.3 Numerical simulations 57 3.3.4 Solid-state NMR 60 3.4 Discussio 66 3.5 Conclusion 68 3.6 References 68 Chapter 4 Transformation of Octacalcium Phosphate to Hydroxyapatite: A Molecular Mechanism Study 70 4.1 Introduction 70 4.2 Experimental section 73 4.3 Results and Analyses 76 4.3.1 SEM 76 4.3.2 TEM and ED 78 4.3.3 XRD 80 4.3.4 Solid-State NMR 82 4.3.5 Computer Assisted Lattice Matching 94 4.4 Discussion 97 4.5 Conclusion 103 4.6 References 104 Chapter 5 Solid-State NMR Study of Rat Dentine 107 5.1 Introduction 107 5.2 Experimental section 109 5.2.1 Biological Samples 109 5.2.2 Characterization 110 5.3 Results 111 5.3.1 SEM& EDX 111 5.3.2 FT-IR 112 5.3.3 Solid-state NMR 113 5.4 Discussions 124 5.5 Conclusion 125 5.6 References 126 Chapter 6 Conclusion and Outlook 128 Appendix A 131 Appendix B 13