Fabrication And Evaluation Of Hydroxyapatite-Chitosan Scaffold Using Simulated Body Fluid Method

ObjectivesThis research aims to fabricate a hydroxyapatite-chitosan scaffold using SBF biomimetic mineralization method and evaluate the physical and chemical properties, water absorption capacity, swelling capacity and degradation property of the scaffold, after that we also study how the mineralization time affects the construction of the scaffolds and seed the osteogenic-induced ADSCs into the scaffolds,we observe it’s proliferation and differentiation capacity to identify the scaffold’s cytocompatibility so that we can understand how the scaffold influence cell growth further more.MethodsThe chitosan powder dissolved in acetic acid solution,we fabricated chitosan scaffolds using the freeze-drying technique, NaOH ethanol aqueous solution was carried to remove the residual acid, cross-linked with sodium tripolyphosphate solution,we put the scaffolds into the progress of precalcification.We put the precalcificated scaffolds into the SBF to conduct the mineralization process and set the mineralization time of 7,14 and 21 days, namely the three experimental groups. Pure chitosan scaffolds acted as the control group.We investigated the scaffold’s physicochemical property. The osteogenic-induced ADSCs were seeded into the scaffolds to investigate the scaffold’s adhesion ability, Live/Dead staining, Type I collagen staining and Alizarin red staining were applied to investigate its properties. Quantitative analysis were used to confirm the cell proliferation and the ALP activity.Results1. The mineral substance of the chitosan scaffolds in simulated body fluid has a good morphology with the matrix in 14th day.2. The mineral substance of 14 days group had a uniform distribution. The mineral substance’s crystal composition suited the HA’s features.3. When the mineralization time is insufficient, the complex mineralization of chitosan scaffold is carbonated hydroxyapatite, when the mineralization time is long enough, the composite mineralization of the scaffold is phosphated hydroxyapatit.4. The compressive/tensile elastic modulus increased with the extension of the mineralization time. When the mineralization time reached at the 21 days, The 21 days group had a statistically significant increase of the compressive/tensile elastic modulus, comparing with control group (P<0.05).5. When the scaffold begun to degrade in the degradation solution, its degradation rate increases fast, with the extension of degradation time, the degradation rate of the scaffold stays stable, the pH of the degradation solution increased within the first week and decreased within the second week, in the subsequent period of time the pH rised slowly until reached at 7.54.6. The 14 days group had the best adhesion and bioactive properties, it also had the most cell proliferation quantitation and was significantly higher than the other three groups(P<0.05).7. With the increasing of cell culture time, the cells seeded on the 14 days group had the most type Ⅰ collagen and the calcified nodules.Conclusions1. The physical and chemical properties achieve the best state in the fourteenth day when the scaffold made by SBF biomimetic mineralization method.2. With the increasing of cell culture time,the water absorption rate increased,the swelling rate decreased,degradation rate and pH tends to be stable.3.ADSCs can make good osteogenic differentiation under the condition of the existence of osteogenic induction medium,Dexamethasone is the main inducer,it can improve the activity of ALP in cells significantly.4.After the 14 days group inoculated with the cells,it had a good adhesion ability, active growth and high proliferation rate,it’s alkaline phosphatase activity was good too. The biocompatibility of the cell scaffold complex was the strongest in the 14 day group,the scaffold of 14 days mineralization time can provide the strongest support to the cell’s differentiation when the cell differentiation medium was sufficient.5.The SBF biomimetic mineralization method is a novel tissue engineering method, it can be used to fabricate HA-chitosan bone tissue engineering scaffolds.