Study On Optimization And Biocompatibility Evaluation Of Multi-component Nano-hydroxyapatite-based Biomaterials

Objective Prepare five group materials with different proportion of the new multi-component nano-based hydroxyapatite two-dimensional composite material, MC3T3-E1 cells were co-cultured with the five groups. Optimize the best adhesion and proliferation group by the inverted phase contrast microscope, scanning electron microscopy, methyl thiazolyl tetrazolium assay, F-DA staining. The three-dimensional scaffold was constructed according to the third group. Ealuating the cell compatibility and tissue compatibility of the three-dimensional scaffold to provide a theoretical basis for design and preparation of the biological materials, and a technique support for broadening the clinic appliance of biomaterial.Methods1. Optimizing two-dimensional materials: Culturing the MC3T3-E1 cells in vitro. The cells in good condition are chosen for experiment. The groups are designed as follows:1 (material1+cells); 2 (material2+cells); 3 (material3+cells); 4 (material4+cells); 5 (material5+cells); 6 blank group(cells + medium).The attachment, growth and proliferation of MC3T3-E1 cells are detected through the inverted phase contrast microscope, scanning electron microscopy ,methyl thiazolyl tetrazolium assay, F-DA staining.2. Cell compatibility of three-dimensional composite scaffold:The groups are designed as follows: material group(material + cells + medium) blank group (cells + medium)The attachment, growth and proliferation of MC3T3-E1 cells are detected through the inverted phase contrast microscope, HE staining ,methyl thiazolyl tetrazolium assay, scanning electron microscopy, F-DA and Hoechst33258 staining.3. Tissue compatibility of three-dimensional composite scaffold: The group was designed as follows: material group (material+ Sprague-Dawley rat), blank group (Sprague-Dawley rat). Observe the inflammation response and vascularization of composite scaffold in Sprague-Dawley rat.Results1Culturing MC3T3-E1 cells in vitro Under the inverted phase contrast microscope:Just seeded in the culture flask, the passage cells are round and in suspension condition. After 2h~3h the cells begin to adhere , after 24h the cells look spindle,polygonal or spindle-shaped and protrude out the synapses connected with neighboring cells, after 48h the cells gradually connect and become into sheet condition, after 3~5d cells become compact single layer and ill-defined.2 Two-dimensional materials group1) Under the inverted phase contrast microscope, the materials are opaque, the cells'growth condition on the materials can not be observed.2) SEM observation shows that the shape of cells on the two-dimensional materials is changing as the time goes on. By 14d the shape cells on the surface of the third group were very full.3) The MTT assay demonstrates that the third group have the best adhesion property, the appreciation rate of cells on the third group is obviously higher than on the fifth group (P<0.05), and similar with the blank group. (P>0.05)4) F-DA staining observation shows that the cells on the surface of the third group grow best and aggregately, and have a tendency to form nodules.3 The cells compatibility of three-dimensional material1) The cells grow better in the composite scaffold by H E staining.2) SEM observation shows that the shape of cells on the composite scaffold is very saturated, like spindle or polygonal .The cells spread multiple filament processes, forming an anchor-like structure, tightly adhere to the material and have a good spread condition.3) F-DA staining observation shows that the cells grow better ,look fusiform or polygonal, aggregate growth in some region and have a tendency to form nodules.4) Hoechst33258 staining observation shows that the cells grow not only on the surface of composite scaffold but also in the pore of the composite scaffold, become multi-layer growth.5) The MTT assay demonstrats that there is no significant difference in cells viability between the material and blank groups (P>0.05) at different time.4 The tissue compatibility of three-dimensional material subcutaneous embedding of Sprague-Dawley ratThere is formation of blood vessel in the composite scaffold and the response of inflammation was mild.Conclusion Multi-component nano-hydroxyapatite-based biomaterials have good biocompatibility, in vitro they promote adhesion and proliferation of MC3T3-E1 cells, in vivo there are mild inflammation and formation of blood vessel in the composite scaffold. The Multi-component nano-hydroxyapatite-based biomaterial will be an extremely promising material in the bone tissue engineering.