A Study Of Osteoinduction Mechanism Of Hydroxyapatite Based On Biomics

Currently, one of the main difficulties in repairing bone defects is the limited availability of suitable biomaterials capable of filling the defect and promoting bone growth. Hydroxyapatite (HA), the main inorganic component of bone, has been widely used in human bone tissue restoration due to its good biocompatibility, osteoconductive performance and osteoinductive properties. There are two main methods of obtaining HA:synthesis by chemical methods, the product of which is defined as synthetic hydroxyapatite (SHA); extraction from natural tissues such as bone, teeth and scales, the product of which is defined as natural hydroxyapatite (NHA). Synthetic hydroxyapatite is a stoichiometric HA with a chemical composition of Ca10(PO4)6(OH)2. Whereas, besides the composition of HA, natural hydroxyapatite also includes trace amounts of ions which reside in the inorganic component of bone, thus could achieve maximum similarity with natural bone mineral. Nowadays, NHA-and SHA-based medical biomaterials are widely used in bone repair. However, comparative studies of the physicochemical and biological properties of NHA and SHA are still lacking. In particular, high-throughput techniques such as proteomics technique have not yet to be used to compare the bone-repair properties of NHA and SHA.In our group’s previous study, natural and synthetic hydroxyapatites were prepared and characterized, and the protein adsorptive properties of NHA and SHA were compared. In addition, NHA-induced osteogenic differentiation of mesenchymal stem cells (MSCs) was studied using transcriptomic technology combined with bioinformatics analysis. Based on our group’s previous research, the present study will compare systematically natural and synthetic hydroxyapatite, including physicochemical and biological properties, the ability to promote bone repair and their mechanism. Then the NHA-induced osteogenic differentiation pathway and the regulatory function of miRNA in those pathways were studied through an integrated study using transcriptomics, proteomics and miRNA analyses. This work consists of two parts.The first part is the comparative study of physicochemical and biological properties of natural and synthetic hydroxyapatite. The main contents are as follows.1) The powdered and disc-shaped samples of both NHA and SHA were prepared, and the physicochemical properties of the samples were characterized. The results revealed that NHA, rather than SHA, contains carbonate and hydrogen phosphate groups, as well as elements such as Mg and Na. NHA exhibited larger single crystal size and higher crystallinity than SHA. In addition, the detection of Ca ion in the extracts implied that NHA exhibits greater physical stability and that SHA might display higher dissolution-deposition. The detection of Mg ion in the extracts implied Mg ion was released only from NHA.2) Mouse bone marrow derived MSCs were isolated using density gradient centrifugation and adherence screening methods. Then flow cytometric analysis was performed to evaluate cell surface markers and MSCs homogeneity. The results showed that the tested fourth-passage cell samples typically expressed the cell adhesion molecules CD29 and CD44 but were negative for the typical lymphocytic marker CD 14 and hematopoietic marker CD34. These results suggested that fourth-passage MSCs were well-purified, and the cultured cells were indeed MSCs.3) The morphology and proliferation of MSCs cultured on both NHA and SHA were investigated using fluorescent staining and MTT assay. The results showed that MSCs cultured on NHA and SHA displayed similar morphology. Compared with the negative control, cells cultured on both NHA and SHA tended to aggregate during growth and proliferation rates were lower.4) The protein samples of cells cultured on both NHA and SHA were labeled with iTRAQ and then subjected to proteomics using 2-D strong cationic exchange/reversed phase liquid chromatography matrix-assisted laser desorption/ionization-tandem mass spectrometry. The results showed that a total of 800 credible proteins were identified in both NHA and SHA at 24 and 72 h. In addition, western blot verification showed the expression levels of the tested proteins were found to be generally consistent between the western blot and proteomics results, which demonstrates the reliability of proteomics data in this study.5) Proteomics data were further subjected to the bioinformatics analyses. The results showed that natural and synthetic hydroxyapatites similarly affect GO functional categories, whereas there are some different functions such as "response to inorganic substance", "ion transmembrane transport", "biomineral formation" and "blood vessel development" in NHA and SHA groups. In addition, NHA and SHA could have induced changes in pathways mediating proliferation, adhesion and differentiation. Also, the biological pathways could able to influence each other through protein interaction network.6) The mineralization of cells cultured on disc-shaped NHA and SHA samples was evaluated. The results showed that the degree of mineralization in NHA and SHA was slightly higher than that of the control group. However, NHA exhibited a higher capacity for bone mineralization than SHA.The second part is an integrated analysis of natural hydroxyapatite-induced osteogenic differentiation of mesenchymal stem cells using transcriptomics, proteomics and microRNA analyses. The main contents are as follows.1) It was compared that the proteomics and gene expression profiling data of MSCs cultured on NHA. The results showed that the numbers of up-regulated genes or proteins are greater than the number of down-regulated in the two groups at each time point. Through the GO categories analysis for proteomics data,4 functional nodes associated with osteogenic differentiation were filtered, including skeletal development, regulation of cell differentiation, negative regulation of cell differentiation and positive regulation of cell differentiation. The 4 functional nodes were included in the 13 functional nodes associated with osteogenic differentiation in gene expression profiling data.10 proteins related to NHA osteogenic differentiation was found involved in these 4 functional nodes.2) By integrating transcriptomics and proteomics, the mRNAs and proteins involved in 89 pathways were shown to be differentially expressed. Among them, some pathways such as MAPK and TGF-beta Receptor pathway might play an important role in NHA-induced osteogenic differentiation.3) The regulatory functions of miRNAs in natural hydroxyapatite-induced osteogenic differentiation were analyzed. Among detected 13 miRNAs,9 miRNAs were participated in regulating differentiation-related biological pathways. In which, miR-26a and miR-26b might inhibit adipogenesis through regulating adipogenesis pathway; miR-222 might play an important role in promoting osteogenic differentiation through regulating MAPK signaling pathway.4) The osteoinduction potential of natural hydroxyapatite was evaluated. The ALP staining showed that MSCs cultured on NHA could be induced to osteogenic differentiation. A verification experiment for the osteogenic pathway indicated that both the ERK1/2 and JNK pathways were responsible for NHA-induced osteogenic differentiation, but the ERK1/2 pathway might be predominant.