Study On The Osteogenic Properties Of Silk Fibroin/Octacalcium Phosphate Compound Porous Scaffolds

Part Ⅰ: Preparation and Characterization of Silk Fibroin / Octacalcium Phosphate CompoundObjective: To select HA precursors of octacalcium phosphate(OCP) and silk fibroin(SF) compound with better osteogenic properties; To adopt homogeneous precipitation method for the preparation of silk fibroin / octacalcium phosphate(SF/OCP) compound materials; To study the relationship between the correlation and response of SF concentration, temperature, reaction time and the formation of OCP; and To study the surface characteristics of SF/OCP compound materials.Methods: mulberry silk was taken and mixed into the aqueous solution containing sodium carbonate and boiled, stirred and scrubbed, and then dried in oven. Then, it was dissolved in lithium bromide solution and stirred, filtered, concentrated and centrifuged. SF was then obtained as the supernatant fluid. The solution of Na H2PO4 and Ca(CH3COO)2 containing following concentrations of SF was prepared: 0, 0.1%, 0.3%, 1%, 2% and 4%, wt/v%. Then, during stirring, Na H2PO4 solution was added and allowed the reaction for 0.5h. Then, the centrifuging precipitate was washed under normal p H. Finally, the precipitate was freeze-dried to obtain SF/OCP complex pink powder. Then, according to the concentration of SF in calcium solution(0%, 0.1%, 0.3%, 1%, 2%, and 4% wt/v%), six products: OCP, 0.1SF/OCP, 0.3SF/OCP, 1SF/OCP, 2SF/OCP and 4SF/OCP were prepared. Then, the characterization was observed and measured through FTIR, XRD, SEM and TEM.Results: through the analysis of FTIR and XRD observations relative to pure OCP or SF, OCP and SF complex have shown characteristic absorption bands in infrared spectrum. SF/OCP compounds prepared under different conditions showed characteristic peaks of OCP, and characteristics peak size varies with SF content. Pure OCP under SEM and TEM scanning or transmission manifested as typical thin flake(lamellar) crystals and in the presence of SF, SF/OCP compound preparation conditions, OCP also showed the typical morphology of OCP crystal.Conclusion: Successful preparation of OCP crystals and SF/OCP compounds was confirmed through FTIR, XRD, SEM and TEM observation and detection. The introduction of SF can regulate the OCP formation, especially smaller sized OCP crystals. Part Ⅱ: Study on Silk Fibroin / Octacalcium Phosphate Compound Porous Scaffold Preparation and PropertiesObjective: To understand the changes in material composition and physiochemical properties of the solution, inside in-vitro simulated body fluid or cell culture process. To investigate the transformation process of OCP to HA. To observe adhesion, growth, proliferation, and in-vitro toxicity of osteoblasts over the materials prepared under different conditions. To study the cytocompatibility problems of the materials and the relationship between performance and preparation conditions of size of pore and dynamics etc. of SF/OCP porous scaffolds.Methods: First, SF/OCP compound material was prepared, and 5mg and 10 mg of SF/OCP compounds were mixed uniformly with 10% SF solution. Then, freeze dried to obtain porous scaffolds. Then, soaked in methanol at room temperature for 1 hour and washed with water and then freeze dried. According to the difference in contents, porous scaffolds containing 5mg and 10 mg SF/OCP compounds were tagged as SF/OCP-1 and SF/OCP-2 complex porous scaffolds respectively. Again, the characterization of SF/OCP compound porous scaffolds was observed through SEM, TEM and TGA. Then, three samples, including SF, SF/OCP-1 and SF/OCP-2 were prepared, immersed in deionized water, and then, the collapsibility, cell compatibility and mechanical properties of the samples at different time points were observed and recorded. Then, prepared samples of SF, SF/OCP-1 and SF/OCP-2 underwent cell culture. Then, light absorption value of each pore was measured by using enzyme linked immunosorbent assay(OD) and results were recorded. Then, MC3T3-E1 cells were ingested over SF, SF/OCP-1 and SF/OCP-2 samples and placed in the incubator for 2, 4, 6 days. Then, CCK-8 reagent was added in different time points and OD values were determined to understand the cellular compatibility.Results: In SF/OCP compounds, OCP showed the typical morphological features of OCP crystals. i.e. a single thin flaky crystal formation. The diameter of a crystal was about 100 to 300 nm. SF/OCP complex elemental analysis further validated that that the crystal formed in SF/OCP complex is OCP crystal. In SF/OCP compound porous scaffolds prepared in pure SF solution, pores were uniformly distributed and the diameter was about 30 to100 nm. In SF/OCP-1 compound porous scaffolds, pores were irregularly distributed and larger compared to that obtained in SF/OCP compound scaffolds and diameter was about 30 to 200 nm. In SF/OCP-2 compound scaffolds, pores were irregularly distributed and smaller compared to that obtained in SF/OCP compound scaffolds. In extract toxicity experiment of SF, SF/OCP-1 and SF/OCP-2 samples, cell relative growth rate(RGR) was higher than 100% and cell toxicity grading(CTS) was 0 which proved good cellular compatibility and no toxic effects on cell growth. Pure OCP cannot be easily broken, thus forming the experiment, combined with SF to improve the mechanical properties. This experiment will freeze drying after SF/OCP compound scaffold further adopts methanol, obtained by this method SF/OCP compound scaffolds in water with good anti washout property.Conclusion: addition of different quantities of SF/OCP compounds can influence the basic morphology and pore size of the scaffolds. The porosity and pore size are the basic conditions essential for the cell growth inside the scaffolds. SF/OCP porous scaffolds have higher ordered porous hierarchical structure, better pore size, larger surface area, and better biological performance. Cell activity is not influenced by SF/OCP compound materials under cell biocompatibility and in-vitro toxicity tests. The SF/OCP compound scaffolds obtained in this experiment through methanol post-processing has better anti-washout property. Part Ⅲ : Study on Silk Fibroin / Octacalcium Phosphate Compound Porous Scaffolds in Vivo OsteogenesisObjective: In the view of present tissue engineering, bone repair ability of the bone scaffold materials is not an ideal problem. Selection of HA precursors of OCP and porous scaffolds obtained from SF compound with good osteogenic properties, in order to improve the bone repairing effect. To focus on the transformation of OCP to HA in-vivo inside the compound scaffolds, and regulation of osteoblasts and osteoclasts and synthesis of bone matrix on OCP and degradation of OCP thereby regulating osteogenesis. This study intends to investigate the osteogenic mechanism of the materials, to provide theoretical basis and new ideas for the construction of tissue-engineered bone materials.Methods: Healthy male SD rats were taken and anaesthetized. An incision was made at the dorsal aspect of the trunk and dissected layer by layer up to the muscles. Then, SF scaffolds and SF/OCP compound scaffolds were separately implanted on the muscle pouches formed on the left and right sides, respectively. Incisions were closed forming intramuscular preparations. Specimens were taken at 4th post-operative week. After gross examination, the specimen was fixed on formalin and decalcified. Then, sequentially dehydrated, made transparent using toluene and dipped into paraffin solution. Then, thin slice of the specimen was taken and stained with H&E, and the condition of material degradation was observed under light microscope.Again, Healthy male SD rats were taken and anaesthetized. An incision was made along the sagittal axis of rats’ skull to expose parietal bone. Then, rat skull bone defect models, with bone defects on each side of parietal bone, were established by using 5mm ring drill. Then, SF scaffold and SF/OCP compound scaffold were implanted on each defect and incision was closed. Rats were allowed normal feeding post-operatively. After 6 and 12 weeks, implanted materials were taken out and micro-CT examination was done to observe bone formation. Then, the rats were sacrificed and the specimen of parietal bone was taken out. Then, the specimens were fixed on formalin. Later, the specimens fixed on formalin were washed first with PBS solution then with distilled water, and then cleaned and decalcified for a week. After decalcification, the specimens were dipped into paraffin solution and thin slices were made using slicing machine and baked. Finally, all slices were stained with H&E and observed under the light microscope.Results: Through micro-CT examinations and H&E staining conducted on the intramuscular implantation and skull bone defect rat models, the results clearly showed that, the SF/OCP porous compound scaffold had the regulation of osteoblast and synthesis of bone matrix over OCP and degradation of OCP, thereby regulating osteogenesis that promoted the growth of bone tissue.Conclusion: In-vivo implantation of SF/OCP composite porous scaffolds proved that the porous scaffold morphology is beneficial for cell growth and accelerates bone repair process. Osteoblast and bone matrix formations over OCP and degradation of OCP suggest regulation of osteogenesis. The SF/OCP compound porous scaffolds implanted with cells have obvious in-vivo, in-vitro and ectopic osteogenic properties.