Functionalization Of Hydroxyapatite Via Silicate Doping And Mechanism Study On Its Promotive Effect On Bone Mineralization

With development of society, as well as progress of material science, biomedical engineering and so on, simple bioactive materials cannot meet the high requirement of people on biomaterials. Functionalization of existing biomaterials could bring better bioactive and specific functionality to them, which is foundation and guarantee for upgrading biomaterials.Hydroxyapatite (HA) is the major inorganic component of animals'bone. With good biocompatibility HA has been widely used in biomedical field. In this work, we modified HA by silicate doping to prepare silicate doped hydroxyapatite (Si-HA), according to its application in bone defect repair. We then investigated phase compositions, fine structures and biocompatibilities of the materials, explored and proposed mechanism of promotive effect of Si-HA on bone mineralization, as well as pathway of material-cell interaction. The present thesis consists of three parts as follows:1. Preparation, characterization and fine structure analysis of nano Si-HA. A series of nano Si-HA with different Si content were prepared by a wet chemical precipitation method using silicon acetate as Si source. There is no obvious aggregation for prepared nano particles. FTIR spectrums show that Si are incorporated in Si-HA lattice in the form of SiO44-, and OH-are partially lost to retain charge balance in molecule. XRD patterns indicate that Si-HA with low Si content (0-1.6wt%) have a main phase of HA, but higher doping will lead to more impurity phases exist. Sintering at1250℃may produce impurity phase of TCP in Si-HA, and TCP becomes main phase with doped Si increases. Rietveld refinement results suggest that with the increase of Si doping, the length of a-axis increases, while the length of c-axis increases and becomes stable after doped Si reaches0.8wt%. Volume of unit cell of Si-HA increases with the increase of doped Si. The lengths of Si-HA crystal grain in directions parallel to c-axis and vertical to c-axis decrease, which correspond with TEM observations. Microstrains alone above said directions are stable while doped Si within the range of0～1.2wt%, however, microstrain of1.6%Si-HA significantly increases, indicating more impurity phases appear and lattice greatly distorts. Calculated average bond length of P-O increases with doped Si increases, suggesting sites of phosphate in HA are gradually occupied by larger orthosilicate tetrahedrals.2. Biocompatibility and bioactivity evaluation of Si-HA. Si-HA is proved to be a good biomaterial via Ca-P deposition evaluation in vitro. Si doping decreases particle size of crystal grain and accordingly increases grain boundary after being sintered, which provides more nucleation sites for Ca-P deposition and thereby improves bioactivity of the material. In SEM observation and cell (rat bone-marrow mesenchymal stem cells, bMSCSs) proliferation assay, Si-HA exhibit good biocompatibility because viability and proliferation of bMSCs on Si-HA are both well. Si doping do not affect biocompatibility of HA, conversely,0.8%or1.2%Si-HA may enhance proliferation of bMSCs compared to pure HA, thus possessing good biocompatibility and bioactivity. On porous Si-HA scaffold, bMSCs show a good activity. Attachment, growth and migration of the cells demonstrate a good biocompatibility of porous Si-HA scaffold. Extracellular matrix secretion of cells on Si-HA scaffold is much more than the cells on pure HA scaffold, indicating that Si-HA has a beneficial bioactivity of promoting extracellular matrix secretion.3. Mechanism investigation on promotive effect of Si-HA on bone mineralization. Prolyl4-hydroxylase (P4H) is a key enzyme for collagen biosynthesis. The activity of P4H directly influence secretion of collagen. ELISA assay results demonstrate that Si-HA with0.8wt%Si doped is able to enhance P4H activity in bMSCs, indicating Si-doping can accelerate biosynthesis of collagen and therefore improve bone mineralization. Immunohistochemical staining observation further verifies that Si-HA has the ability of enhancing P4H activity in bMSCs. Based on above results, we proposed mechanism of the promotive effect of Si-HA on bone mineralization and related pathway-P4H pathway. In the proposed pathway, orthosilicate ions released from Si-HA get into lumen of rough endoplasmic reticulum and then react with Al3+inhibiting P4H activity to form aluminosilicate, thus relieve inhibition by Al3+. Biosynthesis of collagen will be accelerated and bone mineralization will therefore be improved.