| The purpose of this work was to develop novel 3D coralline-chitosan macroporous composite scaffolds for in vitro bone tissue engineering and investigate cellular responses to these scaffolds. In these composites, coral skeletal material, which is made of calcium carbonate (CaCO3), was simultaneously used as particulate reinforcing phase and gas-forming agent to obtain a structure with large pores and improved mechanical and biological properties. The reaction between the coralline material and the acidic chitosan polymer solvent, which produces carbon dioxide (CO2), was rapidly stopped by the subsequent thermally induced phase separation step, leaving coral particulates in the polymeric structure. Scaffolds containing 5 different proportions of coralline material (0, 25, 50, 75, and 100 wt%) were developed and studied under two different aspects. In a first part, the coralline:chitosan weight ratio parameter was studied for its effects on the physical properties of the scaffolds with a combination of scanning electron microscope (SEM), micro-CT imaging, and compression testing. In a second part, the scaffolds were cultured with mice MSCs. Cellular morphology, DNA content, as well as expression of osteogenic markers alkaline phosphatase (ALP) activity and osteocalcin release were evaluated. The effect of cell culture medium supplementation with beta-glycerophosphate and dexamethasone was studied. The results showed that higher coralline concentration increased the pore wall thickness and favoured large pore formation. Varying the coralline powder to chitosan polymer ratio from 0 to 75 wt% increased the observed pore sizes from 80 microm to 400 microm in average and decreased the porosity from 91% to 78%. The equilibrium compressive modulus was improved proportionally with the coral content, and the 75 wt% composites had a significantly higher modulus than all the other chitosan-based scaffolds. The coralline scaffolds showed by far the highest evaluation of cell number and ALP activity over all the other chitosan-based scaffolds. They were the only materials on which the osteocalcin protein was release throughout the study and generally at a high level. Nevertheless, the coralline:chitosan composite scaffolds containing high coralline ratios generally showed higher results than the pure chitosan scaffolds. Of all the chitosan-based scaffolds, the cells cultured on the 75:25 coralline:chitosan scaffolds obtained the highest peak of ALP activity and generally obtained the highest cell number. While the presence of osteogenic supplements had no obvious effect on cell behaviour and osteogenic differentiation, distinct cell morphology and osteoblastic phenotype expression were observed depending on the coralline to chitosan ratios composing the scaffolds. The results strongly suggest that coralline:chitosan composites, especially those having a high coralline content, may enhance adhesion proliferation and osteogenic differentiation of MSCs in comparison with pure chitosan. In conclusion, composite scaffolds with improved mechanical and biological properties concomitant with large pores were achieved by increasing the coralline:chitosan weight ratio. These composites possess therefore many advantages over coralline and chitosan scaffolds, suggesting that they have an excellent potential as biomatrices for tissue engineering. |
